Channelrhodopsins for optical control of cells

ABSTRACT

The invention, in some aspects relates to compositions and methods for altering cell activity and function and the introduction and use of light-activated ion channels.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/570,429, filed Sep. 13, 2019, which is a continuation of U.S.application Ser. No. 14/357,635, filed May 12, 2014, which is a NationalStage Filing under U.S.C. § 371 of PCT International ApplicationPCT/US12/064665, filed Nov. 12, 2012, which was published under PCTArticle 21(2) in English, which claims benefit under 35 U.S.C. § 119(e)of U.S. Provisional application Ser. No. 61/559,076, filed Nov. 12,2011, the entire content of each of which is incorporated by referenceherein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant Nos. CBET1053233, DMS 0848804, and EFR 10835878 awarded by the National ScienceFoundation, under Contract No. HR0011-12-C-0068 awarded by the DefenseAdvanced Research Projects Agency, and under Grant Nos. DP2 OD002002,R01 DA029639, R01 NS067199, RC1 MH088182 and R01 NS075421 awarded by theNational Institutes of Health. The Government has certain rights in theinvention.

SEQUENCE LISTING

The instant application incorporates by reference the Sequence Listingin the ASCII text file filed May 28, 2020, entitled “MIT-004US(06)ST25.txt”, which file was created on Apr. 16, 2020 the size of whichfile is 53248 bytes.

FIELD OF THE INVENTION

The invention, in some aspects relates to compositions and methods foraltering conductance across membranes, cell activity, and cell function,also relates to the use of exogenous light-activated ion channels incells and subjects.

BACKGROUND OF THE INVENTION

Altering and controlling cell membrane and subcellular region ionpermeability has permitted examination of characteristics of cells,tissues, and organisms. Light-driven pumps and channels have been usedto silence or enhance cell activity and their use has been proposed fordrug screening, therapeutic applications, and for exploring cellular andsubcellular function.

Molecular-genetic methods for preparing cells that can be activated(e.g., depolarized) or inactivated (e.g., hyperpolarized) by specificwavelengths of light have been developed (see, for example, Han, X. andE. S. Boyden, 2007, PLoS ONE 2, e299). It has been identified that thelight-activated cation channel channelrhodopsin-2 (ChR2), and thelight-activated chloride pump halorhodopsin (Halo/NpHR), whentransgenically expressed in cell such as neurons, make them sensitive tobeing activated by blue light, and silenced by yellow light,respectively (Han, X. and E. S. Boyden, 2007, PLoS ONE 2(3): e299;Boyden, E. S., et. al., 2005, Nat Neurosci. 2005 September; 8(9):1263-8.Epub 2005 Aug. 14.). Previously identified light-activated pumps andchannels have been restricted to activation by particular wavelengths oflight, thus limiting their usefulness.

SUMMARY OF THE INVENTION

The invention, in part, relates to isolated light-activated ion channelpolypeptides and methods for their preparation and use. The inventionalso includes isolated nucleic acid sequences that encode light-drivenion channels of the invention as well as vectors and constructs thatcomprise such nucleic acid sequences. In addition, the invention in someaspects includes expression of light-activated ion channel polypeptidesin cells, tissues, and subjects as well as methods for using thelight-activated ion channels to alter conductance across membranes, toalter cell and tissue function, and for use in diagnosis and treatmentof disorders.

The invention, in part, also relates to methods for adjusting thevoltage potential of cells, subcellular regions, or extracellularregions. Some aspects of the invention include methods of incorporatingat least one nucleic acid sequence encoding a light-driven ion channelinto at least one target cell, subcellular region, or extracellularregion, the ion channel functioning to change transmembrane passage ofions in response to a specific wavelength of light. Exposing anexcitable cell that includes an expressed light-driven ion channel ofthe invention to a wavelength of light that activates the channel, mayresult in depolarization of the excitable cell. By contacting a cellthat includes a light activated ion channel of the invention withparticular wavelengths of light, the cell is depolarized. A plurality oflight-activated ion channels activated by different wavelengths of lightin overlapping or non-overlapping pluralities of cells may be used toachieve multi-color depolarization.

In some embodiments, the invention comprises a method for the expressionof newly identified classes of genes that encode light-driven ionchannels, in genetically targeted cells, to allow millisecond-timescalegeneration of depolarizing current in response to pulses of light.Channels of the invention can be genetically expressed in specific cells(e.g., using a virus or other means for delivery) and then used tocontrol cells in intact organisms (including humans) as well as cells invitro, in response to pulses of light. Given that these channels havedifferent activation spectra from one another and from the state of theart (e.g., ChR2/VChR1), they also allow multiple colors of light to beused to depolarize different sets of cells in the same tissue, byexpressing channels with different activation spectra genetically indifferent cells, and then illuminating the tissue with different colorsof light.

In some aspects, the invention uses eukaryotic channelrhodpsins, such aseukaryotic channelrhodpsins, such as Chloromonas subdivisa (alsoreferred to herein as: “ChR87”), Chlamydomonas noctigama (also referredto herein as: “Chrimson” or “Chr88”), and Stigeoclonium helveticum (alsoreferred to herein as: “Chronos” or “ChR90”) rhodopsin, and derivativesthereof, are used to depolarize excitable cells. These channelrhodpsins,or derivatives thereof, can also be used to modify the pH of cells, orto introduce cations as chemical transmitters.

The ability to optically perturb, modify, or control cellular functionoffers many advantages over physical manipulation mechanisms, such asspeed, non-invasiveness, and the ability to easily span vast spatialscales from the nanoscale to macroscale. One such approach is anopto-genetic approach, in which heterologously expressed light-activatedmembrane polypeptides such as a light activated ion channel of theinvention, are used to move ions with various spectra of light.

According to an aspect of the invention, methods of altering ionconductivity of a membrane are provided. The methods including a)expressing in a membrane a light-activated ion channel polypeptidecomprising an amino acid sequence of a wild-type or modifiedlight-activated Chlamydomonas noctigama, Stigeoclonium helveticum, orChloromonas subdivisa polypeptide and b) contacting the light-activatedion channel polypeptide with a light that activates the light-activatedion channel and alters the ion conductivity of the membrane. In someembodiments, the light-activated ion channel polypeptide comprises anamino acid sequence of a wild-type or modified light-activatedChlamydomonas noctigama polypeptide and the activating light has awavelength between 365 nm and 700 nm. In certain embodiments, theactivating light has a wavelength from 530 nm to 640 nm, and optionally,the activating light has a wavelength of 590 nm. In some embodiments,contacting the light-activated ion channel polypeptide with a lighthaving a wavelength greater than 720 nm does not activate the ionchannel. In some embodiments, the membrane is not a membrane in whichthe light-activated ion channel naturally occurs. In some embodiments,the light-activated ion channel is an isolated ion channel. In someembodiments, the membrane is in cell. In some embodiments, the cell is aneuronal cell and the method further comprises contacting the ionchannel polypeptide with a light having a wavelength up to 660 nm underconditions suitable to produce a spike in the neuronal cell. In certainembodiments, the nucleic acid sequence encoding the light-activated ionchannel polypeptide comprises the nucleic acid sequence set forth as SEQID NO:3. In some embodiments, the amino acid sequence of thelight-activated ion channel polypeptide comprises SEQ ID NO:2. In someembodiments, the amino acid sequence of the light-activated ion channelpolypeptide comprises a modified Chlamydomonas noctigama light-activatedion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or99% identity to amino acids 86-320 of SEQ ID NO:2 and 95%, 96%, 97%,98%, 99% or 100% identity to the remaining amino acids in the sequenceset forth as SEQ ID NO:2. In certain embodiments, the amino acidsequence of the light-activated ion channel polypeptide comprises SEQ IDNO:5. In some embodiments, the amino acid sequence of thelight-activated ion channel polypeptide comprises a modifiedChlamydomonas noctigama light-activated ion channel sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids86-320 of SEQ ID NO:5 and 95%, 96%, 97%, 98%, 99% or 100% identity tothe remaining amino acids in the sequence set forth as SEQ ID NO:5. Insome embodiments, the light-activated ion channel polypeptide comprisesan amino acid sequence of a wild-type or modified light-activatedStigeoclonium helveticum polypeptide and the light that activates theion channel has a wavelength between 365 nm and 630 nm. In someembodiments, the light that activates the ion channel has a wavelengthfrom 430 nm to 550 nm, and optionally, has a wavelength of 500 nm. Incertain embodiments, contacting the polypeptide with a light having awavelength greater than 650 nm does not activate the ion channel. Insome embodiments, the cell is a neuronal cell and the method furtherincludes contacting the ion channel polypeptide with a light having awavelength between 430 nm and 550 nm in a manner to produce a spike inthe neuronal cell. In some embodiments, the nucleic acid sequenceencoding the light-activated ion channel polypeptide comprises thenucleic acid sequence set forth as SEQ ID NO:8. In certain embodiments,the amino acid sequence of the light-activated ion channel polypeptidecomprises SEQ ID NO:7. In some embodiments, the amino acid sequence ofthe light-activated ion channel polypeptide comprises a modifiedStigeoclonium helveticum light-activated ion channel sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids61-295 of SEQ ID NO:7 and 95%, 96%, 97%, 98%, 99% or 100% identity tothe remaining amino acids in the sequence set forth as SEQ ID NO:7. Insome embodiments, the light-activated ion channel comprises an aminoacid sequence of a wild-type or modified light-activated Chloromonassubdivisa polypeptide and the light that activates the ion channel is alight having a wavelength of between 365 nm and 630 nm and a peakactivating wavelength of 515 nm. In some embodiments, thelight-activated ion channel is encoded by the nucleic acid sequence setforth as SEQ ID NO:12. In certain embodiments, the amino acid sequenceof the light-activated ion channel is set forth as SEQ ID NO:11. In someembodiments, the amino acid sequence of the light-activated ion channelpolypeptide comprises a modified Chloromonas subdivisa light-activatedion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or99% identity to amino acids 81-315 of SEQ ID NO:11 and 95%, 96%, 97%,98%, 99% or 100% identity to the remaining amino acids in the sequenceset forth as SEQ ID NO:11. In some embodiments, the light-activated ionchannel does not activate in response to contact with light having awavelength greater than 650 nm. In certain embodiments, the membrane isa cell membrane. In some embodiments, the cell is a human cell. In someembodiments, the membrane is a cell membrane of a neuronal cell, anervous system cell, a cardiac cell, a circulatory system cell, a visualsystem cell, or an auditory system cell. In certain embodiments,altering the ion conductivity of the membrane depolarizes the cell.

According to another aspect of the invention, an isolated lightactivated ion channel polypeptide is provided. The light-activated ionchannel polypeptide includes an amino acid sequence of a wild-type ormodified light-activated Chlamydomonas noctigama, Stigeocloniumhelveticum, or Chloromonas subdivisa channel polypeptide. In someembodiments, the light-activated ion channel polypeptide comprises anamino acid sequence of a wild-type or modified light-activatedChlamydomonas noctigama polypeptide and activating the ion channelcomprises contacting the ion channel polypeptide with a light having awavelength between 365 nm and 700 nm. In some embodiments, activatingthe ion channel comprises contacting the ion channel polypeptide with alight having a wavelength from 530 nm to 640 nm, and optionally having awavelength of 590 nm. In some embodiments, contacting the ion channelpolypeptide with a light having a wavelength greater than 720 nm doesnot activate the ion channel. In certain embodiments, the nucleic acidsequence encoding the light-activated ion channel polypeptide comprisesthe nucleic acid sequence set forth as SEQ ID NO:3. In some embodiments,the amino acid sequence of the light-activated ion channel polypeptidecomprises SEQ ID NO:2. In some embodiments, the amino acid sequence ofthe light-activated ion channel polypeptide comprises a modifiedChlamydomonas noctigama light-activated ion channel sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids86-320 of SEQ ID NO:2 and 95%, 96%, 97%, 98%, 99% or 100% identity tothe remaining amino acids in the sequence set forth as SEQ ID NO:2. Insome embodiments, the amino acid sequence of the light-activated ionchannel polypeptide comprises SEQ ID NO:5. In certain embodiments, theamino acid sequence of the light-activated ion channel polypeptidecomprises a modified Chlamydomonas noctigama light-activated ion channelsequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityto amino acids 86-320 of SEQ ID NO:5 and 95%, 96%, 97%, 98%, 99% or 100%identity to the remaining amino acids in the sequence set forth as SEQID NO:5. In some embodiments, the light-activated ion channelpolypeptide comprises an amino acid sequence of a wild-type or modifiedlight-activated Stigeoclonium helveticum polypeptide and activating theion channel comprises contacting the ion channel polypeptide with alight having a wavelength between 365 nm and 630 nm. In someembodiments, activating the ion channel includes contacting the ionchannel polypeptide with a light having a wavelength from 430 nm to 550nm, and optionally having a wavelength of 500 nm. In some embodiments,contacting the ion channel polypeptide with a light having a wavelengthgreater than 650 nm does not activate the ion channel. In certainembodiments, the nucleic acid sequence encoding the light-activated ionchannel polypeptide comprises the nucleic acid sequence set forth as SEQID NO:8. In some embodiments, the amino acid sequence of thelight-activated ion channel polypeptide comprises SEQ ID NO:7. In someembodiments, the amino acid sequence of the light-activated ion channelpolypeptide comprises a modified Stigeoclonium helveticumlight-activated ion channel sequence having at least 70%, 75%, 80%, 85%,90%, 95%, or 99% identity to amino acids 61-295 of SEQ ID NO:7 and 95%,96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in thesequence set forth as SEQ ID NO:7. In certain embodiments, thelight-activated ion channel includes an amino acid sequence of awild-type or modified light-activated Chloromonas subdivisa polypeptideand the light that activates the ion channel is a light having awavelength of between 365 nm and 630 nm and a peak activating wavelengthof 515 nm. In some embodiments, the light-activated ion channel isencoded by the nucleic acid sequence set forth as SEQ ID NO:12. In someembodiments, the amino acid sequence of the light-activated ion channelis set forth as SEQ ID NO:11. In certain embodiments, the amino acidsequence of the light-activated ion channel polypeptide comprises amodified Chloromonas subdivisa light-activated ion channel sequencehaving at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to aminoacids 82-315 of SEQ ID NO:11 and 95%, 96%, 97%, 98%, 99% or 100%identity to the remaining amino acids in the sequence set forth as SEQID NO:11. In some embodiments, the light-activated ion channel does notactivate in response to contact with light having a wavelength greaterthan 515 nm. In some embodiments, the light-activated ion channelpolypeptide is expressed in a membrane. In certain embodiments, themembrane is mammalian cell membrane. In some embodiments, the cell is anexcitable cell. In some embodiments, the cell is in a subject. In someembodiments, the membrane is a cell membrane of a neuronal cell, anervous system cell, a cardiac cell, a circulatory system cell, a visualsystem cell, or an auditory system cell. In certain embodiments,altering the ion conductivity of the membrane depolarizes the cell.

According to another aspect of the invention, a vector that includes anucleic acid sequence that encodes any of the aforementionedlight-activated ion channel polypeptides is provided.

According to another aspect of the invention, a cell that includes anyof the aforementioned light-activated ion channel polypeptides isprovided and the cell is not a cell in which the light-activated ionchannel polypeptide naturally occurs. In some embodiments, the cell is amammalian cell and in certain embodiments the cell is a human cell.

According to another aspect of the invention, methods of assessing theeffect of a candidate compound on ion conductivity of a membrane areprovided. The methods including (a) contacting a test membranecomprising the isolated light-activated ion channel polypeptide of anyone of the aforementioned embodiments with light under conditionssuitable for altering ion conductivity of the membrane; (b) contactingthe test membrane with a candidate compound; and (c) identifying thepresence or absence of a change in ion conductivity of the membranecontacted with the light and the candidate compound compared to ionconductivity in a control cell contacted with the light and notcontacted with the candidate compound; wherein a change in the ionconductivity in the test membrane compared to the control indicates aneffect of the candidate compound on the ion conductivity of the testmembrane. In some embodiments, the membrane is in a cell. In certainembodiments, altering the ion conductivity of the membrane depolarizesthe cell. In some embodiments, a change is an increase in ionconductivity of the membrane. In some embodiments, the change is adecrease in ion conductivity of the membrane.

According to another aspect of the invention, methods of treating adisorder in a subject are provided. The methods include (a)administering to a subject in need of such treatment, a therapeuticallyeffective amount of a light-activated ion channel polypeptide of any oneof the aforementioned embodiments, to treat the disorder and (b)contacting the cell with light and activating the light-activated ionchannel in the cell under conditions sufficient to alter ionconductivity of a cell membrane, wherein altering the conductivity ofthe cell membrane treats the disorder. In some embodiments, altering theion conductivity of the membrane depolarizes the cell.

According to yet another aspect of the invention, methods of performinga 2, 3, 4, 5 or more-color light ion channel activation assay in apopulation of cells are provided. The methods include (a) expressing ablue-light-activated ion channel in a first subpopulation of apopulation of cells; (b) expressing a red-light-activated ion channel ina second subpopulation of the population of cells, wherein the first andsecond subpopulations are non-overlapping subpopulations; (c) contactingthe population of cells with a plurality of blue light test dosescomprising combinations of blue light wavelength, pulse width, andpower; (d) measuring transmembrane voltage deflection in a cell of thesecond subpopulation of cells contacted with the blue light test doses;(e) determining the test blue light dose comprising a maximum blue lightpower that activates the blue-light activated ion channel in firstsubpopulation of cells and results in a minimum sub-thresholdtransmembrane voltage deflection in the second subpopulation of cells;(f) contacting the population of cells with a plurality of blue lighttest doses comprising a lower power than the maximum blue light power of(e); (g) determining the blue light test doses of (f) that activate theblue-light activated ion channel; (h) contacting the population of cellswith a plurality of red light test doses comprising combinations of redlight wavelength, pulse width, and power, (i) determining a red lighttest dose comprising a red light power that activates the secondsubpopulation of cells; and (j) performing an activity assay comprisingcontacting the population of cells with the blue light test dosedetermined in (g) and the red light test dose determined in (i). Incertain embodiments, the plurality of blue light test doses comprisewavelengths, pulse widths, and powers independently selected frombetween 400 nm and 500 nm, 1 ms and 5 ms, and 10 μW/mm² and 1.0 mW/mm²,respectively. In some embodiments, the red light test dose of (i) is thetest dose comprising a minimum red light power that activates the secondpopulation of cells. In some embodiments, measuring the transmembranevoltage deflection in (d) comprises patch clamping a cell of the secondpopulation of cells and determining one or more voltage changes in thecell. In certain embodiments, the determining in (e) comprises alteringthe blue light dose by increasing the blue light power from 0.5 mW/mm²to 10 mW/mm²; and measuring the sub-threshold transmembrane voltagedeflection in the second subpopulation of cells. In some embodiments,the minimum sub-threshold voltage deflection is less than 15 mV, lessthan 10 mV, or less than 5 mV. In some embodiments, the maximum bluelight power in (e) is between 0.4 mW/mm² and 0.6 mW/mm². In someembodiments, the blue light power in (g) is between 50 μW/mm² and 0.4mW/mm². In certain embodiments, the red-light activated ion channelcomprises an amino acid sequence of a wild-type or modifiedlight-activated Chlamydomonas noctigama polypeptide. In someembodiments, the nucleic acid sequence encoding the red light-activatedion channel polypeptide comprises the nucleic acid sequence set forth asSEQ ID NO:3. In some embodiments, the amino acid sequence of the redlight-activated ion channel polypeptide comprises SEQ ID NO:2. In someembodiments, the amino acid sequence of the red light-activated ionchannel polypeptide comprises a modified Chlamydomonas noctigamalight-activated ion channel sequence having at least 70%, 75%, 80%, 85%,90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID NO:2 and 95%,96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in thesequence set forth as SEQ ID NO:2. In certain embodiments, the aminoacid sequence of the red light-activated ion channel polypeptidecomprises SEQ ID NO:5. In some embodiments, the amino acid sequence ofthe red light-activated ion channel polypeptide comprises a modifiedChlamydomonas noctigama light-activated ion channel sequence having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids86-320 of SEQ ID NO:5 and 95%, 96%, 97%, 98%, 99% or 100% identity tothe remaining amino acids in the sequence set forth as SEQ ID NO:5. Insome embodiments, the blue-light activated ion channel comprises anamino acid sequence of a wild-type or modified light-activatedStigeoclonium helveticum polypeptide. In some embodiments, the nucleicacid sequence encoding the blue light-activated ion channel polypeptidecomprises the nucleic acid sequence set forth as SEQ ID NO:8. In someembodiments, the amino acid sequence of the blue light-activated ionchannel polypeptide comprises SEQ ID NO:7. In some embodiments, theamino acid sequence of the blue light-activated ion channel polypeptidecomprises a modified Stigeoclonium helveticum light-activated ionchannel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to amino acids 61-295 of SEQ ID NO:7 and 95%, 96%, 97%, 98%,99% or 100% identity to the remaining amino acids in the sequence setforth as SEQ ID NO:7. In some embodiments, the plurality of red lighttest doses comprise wavelengths, pulse widths, and powers independentlyselected from between 600 nm and 740 nm, 1 ms and 5 ms, and 0.1 mW/mm²and 100 mW/mm², respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B shows a graph of channelrhodopsin photocurrents measured incultured hippocampal neurons. FIG. 1A shows results using red light (660nm) peak photocurrent at 10 mW mm⁻² for is illumination. ChR88 is theonly red light sensitive channelrhodopsin with significant photocurrentat 660 nm. FIG. 1B shows results using blue (4.23 mW mm⁻²) or green(3.66 mW mm⁻²) light peak photocurrent at equal photon flux for 5 msillumination. ChR87, ChR88, and ChR90 all have greater or comparablephotocurrent than ChR2. Solid bar indicates blue light, horizontalstriped bar indicates green light.

FIG. 2 is a graph showing action spectrum at equal photon dose at allwavelengths recorded in HEK293FT cells. ChR2 (470 nm peak) and VChR1(545 nm peak) represent the existing channelrhodopsin color sensitivityrange. ChR87 (515 nm peak) and ChR90 (500 nm peak) are blue green lightsensitive channelrhodopsins. Whereas ChR88 (590 nm peak) is the firstred light sensitive natural channelrhodopsin.

FIG. 3A-B provides example traces of optically-driven spikes in culturedhippocampal neurons. FIG. 3A shows red-light-driven spike trains at lowfrequency for Ch88. Generally ChR88 can reliably drive spikes up to 5Hz. However at higher frequency such as 20 Hz, ChR88 desensitizes and/orcauses depolarization block. FIG. 3B shows green-light-driven spiketrains at high frequency for Ch90. Due to ChR90 fast tau off and peakphotocurrent recovery kinetics, it is able to drive temporally precisespikes at the highest frequency a neuron is capable of mediating.

FIG. 4A-B provides graphs showing channelrhodopsin kinetics measured inhippocampal neuron culture voltage clamped at −65 mV. FIG. 4A showssingle exponential channel turn-off kinetics based on 5 ms pulse. ChR90has the fastest turn-off kinetics (3.5 ms) observed across all naturalchannelrhodopsins. FIG. 4B shows peak photocurrent recovery ratio basedon 1s illumination. ChR87 and ChR90 both have fast peak photocurrentrecovery at around 70%. However ChR88 has slow recovery kinetics similarto ChR2.

FIG. 5A-B provides and graph and traces showing Chrimson blue lightcrosstalk characterization in cultured neurons. FIG. 5A shows actionspectrum of Chrimson and the blue light (470 nm) wavelength used forillumination. Wavelength was chosen to minimize crosstalk. FIG. 5Bprovides representative traces from a single neuron at variousillumination conditions. When the blue light power is doubled from 0.1to 0.2 mW mm⁻² while the stimulation protocol is fixed as 5 ms 5 Hz, thevoltage deflection is also doubled. However when the blue light power isfixed at 0.1 mW mm⁻² but the pulse duration is changed from 5 ms to 1000ms, the crosstalk is changed from <5 mV to full spiking correspondingly.This means blue light crosstalk is a function of both light power andlight pulse duration (total photon count).

FIG. 6A-B provides graphs and traces illustrating Chronos and ChR2 bluelight sensitivity in cultured hippocampal neurons. FIG. 6A is a spikeirradiance curve for individual neurons.

FIG. 6B shows lowest light power needed for single-cell 100% spikeprobability vs GFP fluorescence. Chronos (circles) is approximately 5times more light sensitive than ChR2 (triangles) at a given (GFP)expression level. FIG. 6C provides example traces of Chronos spiking atvarious light powers. FIG. 6D illustrates that controls shows nosignificant electrical differences between ChR2 and Chronos expressingneurons.

FIG. 7A-B provides a graph and photomicrographic images illustrating thestrategy used for slice characterization of Chronos and Chrimson. FIG.7A shows illumination wavelength used for slice experiments. FIG. 7B ismicrographic images showing histology for Chronos and Chrimson GFPfusion construct singly expressed in layer 2/3 visual cortex in mice.

FIG. 8A-C provides graphs illustrating whole cell patch clampcharacterization of Chrimson and Chronos blue and red light sensitivityin slice. FIG. 8A illustrates that red light elicits 100% spiking inChrimson expressing neurons but not Chronos expressing neurons between1-6.5 mW mm⁻². FIG. 8B shows that blue light at 0.2-0.5 mW mm⁻² canelicit 100% spiking in Chronos expressing cells but not Chrimsonexpressing cells. However full spiking crosstalk in Chrimson expressingcells can occur at powers higher than 0.6 mW mm⁻². FIG. 8C shows bluelight crosstalk voltage of Chrimson expressing neurons.

FIG. 9 provides example traces of current-clamped opsin-expressingneurons in layer 2/3 slice blue light 0.1 mW mm⁻², red light 1 mW mm⁻²expressing. No crosstalk was observed under red light for Chronos whileminimal subthreshold (<5 mV) crosstalk was observed under blue light forChrimson.

FIG. 10 provides example traces of voltage-clamped non-opsin-expressingneurons in layer 2/3 or 5, post-synaptic to opsin-expressing cells. Zeropost-synaptic crosstalk was observed for both Chronos and Chrimson underred and blue light illumination respectively.

Chronos: blue light 0.13 mW mm⁻², red light 1.7 mW mm⁻².

Chrimson: blue light 0.37 mW mm⁻², red light 1.7 mW mm⁻².

FIG. 11A-C provides a schematic diagram, photomicrographic image andtraces illustrating paired-pulse illumination in slice thatdifferentially express Chrimson and Chronos in separate neurons. FIG.11A shows a triple plasmid in utero electroporation scheme to obtainnon-overlapping expression of Chrimson and Chronos. FIG. 11B shows opsinexpression in visual cortex no overlap of GFP and m02 is observed ratioof Chronos to Chrimson labeling can be tuned by titrating Cre plasmid.FIG. 11C shows voltage-clamped non-opsin-expressing neuron in layer 2/3paired-pulse stimulation to demonstrate different synapses areselectively driven by blue and red light. blue: 0.2 mW mm⁻²; red: 5 mWmm⁻². Arrows represent light application. First trace from top: firstarrow indicates blue light, second arrow indicates red light; secondtrace from top: first arrow indicates red light, second arrow indicatesblue light; third trace from top: both arrows represent red light; andfourth trace from top: both arrows represent blue light.

FIG. 12 is a trace illustrating that Chrimson can drive spikes in thefar-red (660 nm) using 5 ms pulses at 2.6 mW mm⁻² in culturedhippocampal neurons.

FIG. 13A-C provides traces illustrating that the ChR88 K176R mutant hasimproved kinetics (13 ms tau off) and can mediate high frequency spikesin cultured hippocampal neurons. Exemplar current clamped traces of asingle ChR88 K176R expressing neuron are shown. FIG. 13A shows thatChR88 K176R can reliably drive spikes from 1 to 10 mW mm⁻² at 625 nm 5Hz stimulation. FIG. 13B shows red light (625 nm) driven spike trains atvarious frequency for ChR88 K176R. 1 mW mm⁻² light power is used for allfrequencies. FIG. 13C shows current injection control to show the neuronis capable of spiking at the indicated frequencies.

FIG. 14 provides graphs showing channelrhodopsin ion selectivitymeasured in HEK293FT cells. ChR88 and ChR90 have comparable ionselectivity as ChR2. However ChR87 has less sodium (Na) current comparedto calcium (Ca), proton (H), and potassium (K) current.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is amino acid sequence from Chlamydomonas noctigamaMAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTVTHPTSNLANRNSFVIMAERMRARGIDVRASLDRNGPMIESGRVILADTDIFVTEMFKAQFAQLPAAIELIPALGADNALQLVQQASVLGGCDFVMVHPQFLKDNSPSGLVARLRMMGQRVVAFGPANLRELIESCDVDAWIEAPPINLYQLRQVVAQMQLMRRQAAMMGGMGGGMKGGMSGMGMGMHAGSMWKQQQMMMQQDGSAMMMPAMQGGAASMRGSGLISAQPGRQASLGGPQSVMMGSAMVGSNPLFGTAPSPLGSAVGAEAMGHNLYGNQAAAGGIPAASAAADGTDVEMMQQLMSEIDRLKGELGEQDMPR.SEQ ID NO: 2: ChR88 coding amino acid sequence that includes residues 1-350 ofSEQ ID NO: 1 MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEF VEEEDEDTV.SEQ ID NO: 3 is a mammalian-codon optimized DNA sequence encoding ChR88 light-activated ion channel polypeptideatggctgagctgatcagcagcgccaccagatctctgtttgccgccggaggcatcaacccttggcctaacccctaccaccacgaggacatgggctgtggaggaatgacacctacaggcgagtgatcagcaccgagtggtggtgtgacccttcttacggactgagcgacgccggatacggatattgcttcgtggaggccacaggcggctacctggtcgtgggagtggagaagaagcaggcttggctgcacagcagaggcacaccaggagaaaagatcggcgcccaggtctgccagtggattgctttcagcatcgccatcgccctgctgacattctacggcttcagcgcctggaaggccacttgcggttgggaggaggtctacgtctgttgcgtcgaggtgctgttcgtgaccctggagatcttcaaggagttcagcagccccgccacagtgtacctgtctaccggcaaccacgcctattgcctgcgctacttcgagtggctgctgtcttgccccgtgatcctgatcaagctgagcaacctgagcggcctgaagaacgactacagcaagcggaccatgggcctgatcgtgtcttgcgtgggaatgatcgtgttcggcatggccgcaggactggctaccgattggctcaagtggctgctgtatatcgtgtcttgcatctacggcggctacatgtacttccaggccgccaagtgctacgtggaagccaaccacagcgtgcctaaaggccattgccgcatggtcgtgaagctgatggcctacgcttacttcgcctcttggggcagctacccaatcctctgggcagtgggaccagaaggactgctgaagctgagcccttacgccaacagcatcggccacagcatctgcgacatcatcgccaaggagttttggaccttcctggcccaccacctgaggatcaagatccacgagcacatcctgatccacggcgacatccggaagaccaccaagatggagatcggaggcgaggaggtggaagtggaagagttcgtggaggaggaggacgaggacacagtgSEQ ID NO: 4 is transmembrane region of ChR88 including residues 86-320 of SEQ ID NO: 2GTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKI HEHILIH.SEQ ID NO: 5 is derived from ChR88 and includes K176R substitutionMAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEF VEEEDEDTV.SEQ ID NO: 6 is amino acid sequence from Stigeoclonium helveticumMETAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGVNHGTADLAHRASFQKMGDRLRAQGVTVRASLDAHEVPPADEENKFAQKSAAANMPAYNPGKVILIVPDMSMVDYFRDQFEQLPTRMELLPALGMDT.SEQ ID NO: 7 is ChR90 coding amino acid sequence that includes residues 1-325 ofSEQ ID NO: 6 METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.SEQ ID NO: 8 is a mammalian-codon optimized DNA sequence encoding ChR90 light-activated ion channel polypeptideatggaaacagccgccacaatgacccacgcctttatctcagccgtgcctagcgccgaagccacaattagaggcctgctgagcgccgcagcagtggtgacaccagcagcagacgctcacggagaaacctctaacgccacaacagccggagccgatcacggttgcttcccccacatcaaccacggaaccgagctgcagcacaagatcgcagtgggactccagtggttcaccgtgatcgtggctatcgtgcagctcatcttctacggttggcacagcttcaaggccacaaccggctgggaggaggtctacgtctgcgtgatcgagctcgtcaagtgcttcatcgagctgttccacgaggtcgacagcccagccacagtgtaccagaccaacggaggagccgtgatttggctgcggtacagcatgtggctcctgacttgccccgtgatcctgatccacctgagcaacctgaccggactgcacgaagagtacagcaagcggaccatgaccatcctggtgaccgacatcggcaacatcgtgtgggggatcacagccgcctttacaaagggccccctgaagatcctgttcttcatgatcggcctgttctacggcgtgacttgatatccagatcgccaaggtgtatatcgagagctaccacaccctgcccaaaggcgtctgccggaagatttgcaagatcatggcctacgtcttcttctgctcttggctgatgttccccgtgatgttcatcgccggacacgagggactgggcctgatcacaccttacaccagcggaatcggccacctgatcctggatctgatcagcaagaacacttggggcttcctgggccaccacctgagagtgaagatccacgagcacatcctgatccacggcgacatccggaagacaaccaccatcaacgtggccggcgagaacatggagatcgagaccttcgtcgacgaggaggaggagggaggagtg.SEQ ID NO: 9 is transmembrane region of ChR90 including residues 61-295 of SEQ ID NO: 7GTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIH SNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHIL IH.SEQ ID NO: 10 is amino acid sequence from Chloromonas subdivisaMSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGAVVPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCPVILIHLSNLTGMKNDYNKRTMALLVSDVGCIVWGTTAALSTDEVKIIFFELGLLYGEYTEYAAAKIYIEAYHTVPKGICRQLVRLQAYDEFFTWSMFPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHEHILVHGNITKKTKVNVAGDMVELDTYVDQDEEHDEGTIDRGTQELANRHSFVVMRENMRAKGVDVRASLGDIDGTEMTKAGNMNGTLEPGRIILCVPDMSLVDFFREQFSQMPVPFEVVPALGPEVALQLVQQALSIGGANY IDYVM.SEQ ID NO: 11 ChR87 coding amino acid sequence that includes residues 1-346 of SEQ IDNO: 10 MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGAVVPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCPVILIHLSNLTGMKNDYNKRTMALLVSDVGCIVWGTTAALSTDEVKIIFFELGLLYGEYTEYAAAKIYIEAYHTVPKGICRQLVRLQAYDEFFTWSNIFPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHEHILVHGNITKKTKVNVAGDMVELDTYVDQ DEEHDEG.SEQ ID NO: 12 is a mammalian-codon optimized DNA sequence encoding ChR87 light-activated ion channel polypeptideatgagcagactggtcgccgcttcttggctgctggctctcctcctctgcggaattaccagcacaacaacagcctctagcgccccagcagcttcttctacagacggaacagccgccgcagcagtgtctcactacgccatgaacggcttcgacgagctggctaaaggagccgtggtgccagaagaccactttgtctgcggaccagccgacaagtgctattgctccgcttggctgcacagccacggaagcaaggaggagaagaccgccttcaccgtcatgcagtggatcgtgttcgccgtctgcatcatcagcctgctgttctacgcctaccagacttggagggctacttgcggttgggaggaggtgtacgtgaccatcatcgagctggtccacgtctgcttcggactctggcacgaggtcgatagcccttgtaccctgtacctgagcacaggcaacatggtcctctggctgagatacgccgagtggctgctgacttgccccgtgatcctgatccacctgagcaacctgaccggcatgaagaacgactacaacaagcggaccatggccctgctggtgtcagacgtgggctgtatcgtgtggggaacaacagccgccctgagcaccgatttcgtgaagatcatcttcttcttcctgggcctgctgtacggcttctacaccttctacgccgccgccaagatctacatcgaggcctaccacaccgtgcccaagggcatttgtagacagctcgtgcggctgcaggcctacgacttcttcttcacttggagcatgttccccatcctgttcatggtcggcccagagggattcggcaagatcaccgcctacagcagcggaatcgcccacgaagtgtgcgatctgctgagcaagaacctctggggcctgatgggccacttcatccgcgtgaagatccacgagcacatcctggtgcacggcaacatcaccaagaagaccaaggtcaacgtggccggcgacatggtggaactggacacctacgtggaccaggacgaggaacacgacgaggga.SEQ ID NO: 13 is transmembrane region of ChR87 including residues 81-315 of SEQ IDNO: 11 GSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCPVILIHLSNLTGMKNDYNKRTMALLVSDVGCIVWGTTAALSTDEVKIIFFELGLLYGEYTFYAAAKIYIEAYHTVPKGICRQLVRLQAYDFFFTWSMFPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHE HILVH.SEQ ID NO: 14 amino acid sequence for Neochlorosarcina sp. Rhodopsin. This light-activated ion channel is referred to herein as ChR62.MADFVWQGAGNGGPSAMVSHYPNGSVLLESSGSCYCEDWYTSRGNHVEHSLSNACDWFAFAISVIELVYYAWAAENSSVGWEEIYVCTVELIKVSIDQFLSSNSPCTLYLSTGNRVLWIRYGEWLLTCPVILIHLSNVTGLKDNYSKRTMALLVSDIGTIVFGVTSAMCTGYPKVIFFILGCCYGANTFFNAAKVYLEAHHTLPKGSCRTLIRLMAYTYYASWGMFPILFVLGPESFGHMNMYQSNIAHTVIDLMSKNIWGMLGHFLRHKIREHILIHGDLRTTTTVNVAGEEMQVETMVAAEDADETTV.SEQ ID NO: 15 is the mammalian codon-optimized DNA sequence for the Neochlorosarcinarhodopsin. This light-activated ion channel is referred to herein as ChR62.atggccgacttcgtgtggcagggagctggaaacggaggaccaagcgccatggtgtcccactaccccaatggcagcgtgctgctggagagctccggcagctgctactgtgaagactggtatacttctcggggcaaccacgtggagcattctctgagtaatgcttgcgattggttcgcctttgctatcagcgtgattttcctggtgtactatgcctgggccgcttttaactctagtgtgggctgggaggaaatctacgtgtgcaccgtggagctgatcaaggtgagcattgatcagttcctgagctccaactctccttgtaccctgtacctgagtacagggaatagggtgctgtggatcagatatggcgaatggctgctgacttgtccagtgatcctgattcacctgtccaacgtgacagggctgaaggacaattactctaaacgcactatggctctgctggtgagtgatatcgggaccatcgtgttcggcgtgacttctgccatgtgcaccggataccccaaagtgatcttctttattctgggctgctgttatggagctaacacattctttaatgccgctaaggtgtacctggaggcccaccatacactgcctaaaggctcttgtaggactctgatcagactgatggcctatacctactatgctagttggggaatgttccccattctgtttgtgctgggacctgagagatcggccacatgaacatgtaccagtccaatatcgcccataccgtgattgacctgatgtccaagaacatctggggaatgctggggcactttctgcggcataaaattcgcgagcacatcctgattcatggagatctgcggaccacaactaccgtgaatgtggctggggaggaaatgcaggtggaaacaatggtggccgctgaggacgccgatgaaacaactgtg.SEQ ID NO: 16 is the amino acid sequence for Heterochlamydomonas inaequalis rhodopsin.This light-activated ion channel is referred to herein as ChR93.MGGIGGGGIQPRDYSYGANGTVCVNPDVCFCLDWQQPFGSNMENNVSQGFQLFTIALSACILMFYAYEWYKATCGWEEIYVCVVEMSKICIELVHEYDTPFCLYLATGSRVLWLRYAEWLMTCPVILIHLSNITGLGTDYNKRTMVLLMSDIGCIVFGATAAFANEGYVKCACFLLGMAWGMNTFYNAAKVYYESYVLVPSGICKLLVAVMAGLYYVSWSLFPILFAIGPEGFGVISLQASTIGHTIADVLSKNMWGLMGHFLRVQIYKHILLHGNIRKPIKLHMLGEEVEVMALVSEEGEDTV.SEQ ID NO: 17 is the mammalian codon-optimized DNA sequence for theHeterochlamydomonas inaequalis rhodopsin, this light-activated ion channel is referred toherein as ChR93.atgggaggaattggcggaggcggcattcagcctagagactacagctacggcgccaacggaacagtctgcgtgaaccccgacgtctgcttctgtctggattggcagcagcccttcggctctaacatggagaacaacgtgtcccagggcttccagctgtttaccatcgccctgagcgcctgcatcctgatgttctacgcctacgagtggtacaaggccacttgcggttgggaggagatctacgtctgcgtggtggagatgagcaagatttgcatcgagctggtgcacgagtacgacacccccttttgcctgtacctggccaccggcagcagagtcctctggctgagatacgccgagtggctcatgacttgccccgtgatcctgatccacctgagcaacatcaccggactgggcaccgactacaacaagcggaccatggtgctcctgatgagcgacatcggttgcatcgtgttcggcgccacagcagcattcgccaacgagggctacgtgaagtgcgcttgtttcctgctgggcatggcttggggcatgaacaccttctacaacgccgccaaggtgtactacgagagctacgtgctggtgccctccggaatttgcaagctgctggtggccgtgatggccggactgtactacgtgtcttggagcctgttccccatcctgtttgccatcggcccagagggatttggcgtgatcagcctgcaggccagcaccattggccacacaatcgccgacgtgctgagcaagaacatgtggggcctgatgggccacttcctgcgggtgcagatctacaagcacatcctgctgcacggcaacatccggaagcctatcaagctgcacatgctgggcgaggaggtggaagtgatggctctggtgtccgaggagggagaggataccgtg.SEQ ID NO: 18 is the mammalian codon-optimized DNA sequence that encodes the wild-type Channelrhodopsin-2, (see: Boyden, E. et al., Nature Neuroscience 8, 1263-1268 (2005)and Nagel, G., et al. PNAS November 25, 2003 vol. 100 no. 24 13940-13945), also referred toherein as ChR2:atggactatggcggcgctttgtctgccgtcggacgcgaacttttgttcgttactaatcctgtggtggtgaacgggtccgtcctggtccctgaggatcaatgttactgtgccggatggattgaatctcgcggcacgaacggcgctcagaccgcgtcaaatgtcctgcagtggcttgcagcaggattcagcattttgctgctgatgttctatgcctaccaaacctggaaatctacatgcggctgggaggagatctatgtgtgcgccattgaaatggttaaggtgattctcgagttatttttgagtttaagaatccctctatgctctaccttgccacaggacaccgggtgcagtggctgcgctatgcagagtggctgctcacttgtcctgtcatccttatccacctgagcaacctcaccggcctgagcaacgactacagcaggagaaccatgggactccttgtctcagacatcgggactatcgtgtggggggctaccagcgccatggcaaccggctatgttaaagtcatcttcttttgtcttggattgtgctatggcgcgaacacattttttcacgccgccaaagcatatatcgagggttatcatactgtgccaaagggtcggtgccgccaggtcgtgaccggcatggcatggctgtttttcgtgagctggggtatgttcccaattctatcattttggggcccgaaggttttggcgtcctgagcgtctatggctccaccgtaggtcacacgattattgatctgatgagtaaaaattgttgggggttgttgggacactacctgcgcgtcctgatccacgagcacatattgattcacggagatatccgcaaaaccaccaaactgaacatcggcggaacggagatcgaggtcgagactctcgtcgaagacgaagccgaggccggagccgtg.SEQ ID NO: 19 is the amino acid sequence of the wild-type Channelrhodopsin-2,, (see:Boyden, E. et al., Nature Neuroscience 8, 1263-1268 (2005) and Nagel, G., et al. PNASNov. 25, 2003 vol. 100 no. 24 13940-13945), also referred to herein as ChR2:MDYGGALSAVGRELLEVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLNIFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEEKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAV.SEQ ID NO: 20 is the DNA sequence of the ′ss′ signal sequence from truncated MHC class Iantigen: gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc.SEQ ID NO: 21 is the amino acid sequence of the ′ss′ signal sequence from truncated MHCclass I antigen: MVPCTLLLLLAAALAPTQTRA.SEQ ID NO: 22 is the DNA sequence of the ER export sequence (also referred to herein asER2″: ttctgctacgagaatgaagtg.SEQ ID NO: 23 is the amino acid sequence of the ER export sequence (also referred to hereinas ″ER2″: FCYENEV.SEQ ID NO: 24 is the DNA sequence of KGC, which is a C terminal export sequence fromthe potassium channel Kir2.1: aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt.SEQ ID NO: 25 is the amino acid sequence of KGC, which is a C terminal export sequencefrom the potassium channel Kir2.1: KSRITSEGEYIPLDQIDINV.

DETAILED DESCRIPTION

The invention in some aspects relates to the expression in cells oflight-driven ion channel polypeptides that can be activated by contactwith one or more pulses of light, which results in strong depolarizationof the cell. Light-activated channels of the invention, also referred toherein as light-activated ion channels can be expressed in specificcells, tissues, and/or organisms and used to control cells in vivo, exvivo, and in vitro in response to pulses of light of a suitablewavelength. Sequences from Chlamydomonas such as Chrimson andderivatives thereof, are strongly activated by contact with red light.In addition, light-activated ion channel polypeptides derived fromStigeoclonium rhodopsin sequences, have now been identified andcharacterized as being activated by light having a wavelength between365 nm and 630 nm.

The light-activated ion channels of the invention are ion channels andmay be expressed in a membrane of a cell. An ion channel is an integralmembrane protein that forms a pore through a membrane and assist inestablishing and modulating the small voltage gradient that existsacross the plasma membrane of all cells and are also found insubcellular membranes of organelles such as the endoplasmic reticulum(ER), mitochondria, etc. When a light-activated ion channel of theinvention is activated by contacting the cell with appropriate light,the pore opens and permits conductance of ions such as sodium,potassium, calcium, etc. through the pore.

In some embodiments of the invention, light-activated channels may beused to modify the transmembrane potential (and/or ionic composition) ofcells (and/or their sub-cellular regions, and their local environment).For example, the use of inwardly rectifying cationic channels willdepolarize cells by moving positively charged ions from theextracellular environment to the cytoplasm. Under certain conditions,their use can decrease the intracellular pH (and/or cationconcentration) or increase the extracellular pH (and/or cationconcentration). In some embodiments, the presence of light-activated ionchannels in one, two, three, or more (e.g. a plurality) of cells in atissue or organism, can result in depolarization of the single cell orthe plurality of cells by contacting the light-activated ion channelswith light of suitable wavelength.

Chlamydomonas-Derived Light-Activated Ion Channels

When expressed in a cell, light-activated ion channels of the inventionidentified having a Chlamydomonas light-activated ion channel sequenceor a derivative thereof, can be activated by contacting the cell withlight having a wavelength between about 365 nm and 700 nm, between 530nm and 640 nm, and in some embodiments, a peak activation may occur uponcontact with light having a wavelength of 590 nm. Examples of theselight-activated ion channels include ChR88 (also referred to herein asChrimson), K176R substituted Chrimson sequence such as SEQ ID NO: 5; andfunctional derivatives thereof. In some embodiments of the invention, aChlamydomonas light-activated ion channel is a Chlamydomonas noctigamalight-activated ion channel.

Contacting an excitable cell that includes a Chlamydomonas-derivedlight-activated ion channel of the invention with a light in theactivating range of wavelengths strongly depolarizes the cell. Exemplarywavelengths of light that may be used to depolarize a cell expressing aChlamydomonas-derived light-activated ion channel of the invention,include wavelengths from at least about 365 nm, 385 nm, 405 nm, 425 nm,445 nm, 465 nm, 485 nm, 505 nm, 525 nm, 545 nm, 565 nm, 585 nm; 605 nm,625 nm, 645 nm, 665 nm, 685 nm; and 700 nm, including all wavelengthstherebetweeen. In some embodiments, Chlamydomonas-derivedlight-activated ion channels of the invention have a peak wavelengthsensitivity in of 590 nm, and may elicit spikes up to 660 nm.

In some embodiments of the invention, a Chlamydomonas-derivedlight-activated ion channel, a non-limiting example of which is Chrimsonor a K176R substituted Chrimson set forth as SEQ ID NO:5, can drivetemporally precise spikes with 1-5 ms pulse width at 0.5 mW/mm² to >10mW/mm² in neurons at its optimal wavelength in slice and in cellculture; and can stimulate at frequency up to 10 Hz reliably at itsoptimal wavelength. In some embodiments of the invention, an optimalwavelength for a Chlamydomonas-derived light-activated ion channel isbetween 530 nm and 640 nm. In certain embodiments of the invention, thesubstituted Chlamydomonas-derived light-activated ion channel having anamino acid sequence set forth as SEQ ID NO:5, has a decreased tau offfrom 25 ms to 13 ms, and this K176R mutant can stimulate at frequency upto 60 Hz reliably at optimal wavelength, which may be between 530 nm and640 nm.

Light-activated ion channels of the invention such as ChR88 andderivatives thereof can be used to depolarize excitable cells in whichone or more light-activated ion channels of the invention are expressed.In some embodiments, Chlamydomonas-derived light-activated ion channelsof the invention can be expressed in a sub-population of cells in a cellpopulation that also includes one or more additional subpopulations ofcells that express light-activated ion channels that are activated bywavelengths of light that do not activate a Chlamydomonas-derivedlight-activated ion channel of the invention.

Stigeoclonium-Derived Light-Activated Ion Channels

When expressed in a cell, light-activated ion channels of the inventionidentified having a Stigeoclonium light-activated ion channel sequenceor a derivative thereof, can be activated by contacting the cell withlight having a wavelength between about 365 nm and 630 nm, between 430nm and 550 nm; and in some embodiments, a peak activation may occur uponcontact with light having a wavelength of 500 nm. Examples of theselight-activated ion channels include ChR90 (also referred to herein asChronos) and functional derivatives thereof. In some embodiments of theinvention, a Stigeoclonium light-activated ion channel is aStigeoclonium helveticum light-activated ion channel.

Contacting an excitable cell that includes a Stigeoclonium-derivedlight-activated ion channel of the invention with a light in theactivating range of wavelengths strongly depolarizes the cell. Exemplarywavelengths of light that may be used to depolarize a cell expressing aStigeoclonium-derived light-activated ion channel of the invention,include wavelengths from at least about 365 nm, 385 nm, 405 nm, 425 nm,445 nm, 465 nm, 485 nm, 505 nm, 525 nm, 545 nm, 565 nm, 585 nm; 605 nm,and 630 nm, including all wavelengths therebetweeen. In someembodiments, Stigeoclonium-derived light-activated ion channels of theinvention have a peak wavelength sensitivity in of 500 nm. In someembodiments of the invention, a Stigeoclonium-derived light-activatedion channel can drive temporally precise spikes with 1-5 ms pulse widthat 50 uW/mm² to >10 mW/mm² in neurons at “optimal wavelength” in sliceand cultured cells; and can stimulate at frequency >100 Hz at “optimalwavelength”. As used herein an optimal wavelength for aStigeoclonium-derived light-activated ion channel may be a wavelengthbetween 430 nm and 550 nm.

Light-activated ion channels of the invention such as ChR90 andderivatives thereof can be used to depolarize excitable cells in whichone or more light-activated ion channels of the invention are expressed.In some embodiments, Stigeoclonium-derived light-activated ion channelsof the invention can be expressed in a sub-population of cells in a cellpopulation that also includes one or more additional subpopulations ofcells that express light-activated ion channels that are activated bywavelengths of light that do not activate a Stigeoclonium-derivedlight-activated ion channel of the invention.

Chloromonas-Derived Light-Activated Ion Channels

When expressed in a cell, light-activated ion channels of the inventionidentified having a Chloromonas light-activated ion channel sequence ora derivative thereof, can be activated by contacting the cell with lighthaving a wavelength between about 365 nm and 630 nm, between 450 nm and570 nm; and in some embodiments, a peak activation may occur uponcontact with light having a wavelength of 525 nm. In some embodiments ofthe invention, a Chloromonas light-activated ion channel, a non-limitingexample of which is ChR87, does not exhibit light sensitivity(activation) at wavelengths greater than 650 nm, and can drivetemporally precise spikes with 1-5 ms pulse width at 0.1 mW/mm² togreater than 10 mW/mm² in neurons at its optimal wavelength in bothslice and cell culture. In some embodiments of the invention, aChloromonas light-activated ion channel (such as ChR87) can stimulate atfrequency >60 Hz at its optimal wavelength. In some aspects of theinvention the optimal wavelength for a Chloromonas light-activated ionchannel, a non-limiting example of which is ChR87, is between 450 and570 nm. Examples of Chloromonas light-activated ion channels includeChR87 and functional derivatives thereof. In some embodiments of theinvention, a Chloromonas light-activated ion channel is a Chloromonassubdivisa light-activated ion channel.

Chloromonas-, Chlamydomonas-, and Stigeoclonium-derived light-activatedion channels of the invention permit ion conductance and depolarizationwhen contacted under suitable conditions with an appropriate wavelengthof light. As will be understood by those in the art, the term“depolarized” used in the context of cells means an upward change in thecell voltage. For example, in an excitable cell at a baseline voltage ofabout −65 mV, a positive change in voltage, e.g., up to 5, 10, 15, 20,30, 40, or more millivolts (mV) is a depolarization of that cell. Whenthe change in voltage is sufficient to reach the cell's spike initiationvoltage threshold an action potential (e.g. a spike) results. When acell is depolarized by activating a light-activated ion channel of theinvention with an appropriate wavelength of light, the cell voltagebecomes more positive than the baseline level, and an incoming signalmay more easily raise the cell's voltage sufficiently to reach thethreshold and trigger an action potential in the cell. It has beendiscovered that by contacting a cell expressing a Chlamydomonas-derivedlight-activated ion channel of the invention with light in the rangebetween about 365 nm to about 700 nm, the voltage of the cell becomesless negative and may rise by at least about 20, 30, 40, 50, 60, 70, 80,90, 100, mV (depending on the cell type) thus, depolarizing the cell.Similarly, it has been discovered that by contacting a cell expressing aStigeoclonium-derived light-activated ion channel of the invention withlight in the range between about 365 nm and 630 nm the voltage of thecell becomes less negative by as much as at least 20, 30, 40, 50, 60,70, 80, 90, 100, mV, (depending on cell type). Similarly, it has beendiscovered that by contacting a cell expressing a Chloromonas-derivedlight-activated ion channel of the invention with light in the rangebetween about 365 nm and 630 nm, or between 450 nm and 570 nm thevoltage of the cell becomes less negative by as much as at least 20, 30,40, 50, 60, 70, 80, 90, 100, mV, (depending on cell type). As usedherein, the term “activate” when used in reference to a light-activatedion channel of the invention, means to open the channel making itpermissive to ion conduction through the channel.

Specific ranges of wavelengths of light that in some embodiments of theinvention are useful to activate ion channels of the invention areprovided and described herein. It will be understood that a light ofappropriate wavelength for activation and will have a power andintensity appropriate for activation. It is well known in the art thatlight pulse duration, intensity, and power are parameters that can bealtered when activating a channel with light. Thus, one skilled in theart will be able to adjust power, intensity appropriately when using awavelength taught herein to activate a light-activated ion channel ofthe invention. A dose light that contacts a light-activated ion channelof the invention may be determined based on the wavelength, pulselength, and power of the light that contacts the light-activated ionchannel. Thus, as a non-limiting example, a dose may have a wavelengthof 550 nm, a 4 ms pulse length, and a 0.5 mW/mm² power and another lightdose may have a wavelength of 550 nm, a 3 ms pulse length and a 0.5mW/mm² power. Those skilled in the art will understand methods to selecta dose of light by independently selecting a wavelength, a pulse length,and a power for the light with which a light-activated ion channel ofthe invention is contacted. In some embodiments, wavelength and pulselength may be held steady, and power incrementally increased to examineactivation parameters of a light-activated ion channel of the invention.Similarly, in certain embodiments of the invention may includeincremental wavelength increases while pulse length and power are heldsteady; or incremental pulse length increases while wavelength and powerare held steady. In some embodiments of the invention two or more ofwavelength, pulse length, and power of a light may be incrementallyaltered to examine the effect on activation of a light-activating ionchannel of the invention.

A benefit of a light-activated ion channel of the invention is theability to “tune” the light-activated ion channel's response usingappropriate illumination variables (e.g., wavelength, intensity,duration, etc.) (also referred to herein as dose) to activate thechannel. Methods of adjusting illumination variables are well-known inthe art and representative methods can be found in publications such as:Lin, J., et al., Biophys. J. 2009 Mar. 4; 96(5):1803-14; Wang, H., etal., 2007 Proc Natl Acad Sci USA. 2007 May 8; 104(19):8143-8. Epub 2007May 1, each of which is incorporated herein by reference. Thus, it ispossible to utilize a narrow range of one or more illuminationcharacteristics to activate a light-activated ion channel of theinvention. The expression of light-activated ion channels that areactivated by different wavelengths of light in distinct, separate,subpopulations in a cell population can permit application of differentillumination parameters to the population with an effect ofdifferentially activating the different subpopulations through the usespecific wavelengths of light. Thus, permitting controlled activation ofa mixed population of light-activated channels.

In exemplary implementations, the invention comprises methods forpreparing and using genes encoding light-activated ion channels of theinvention that have now been identified. The invention, in part, alsoincludes isolated nucleic acids comprising sequences that encode lightactivated ion channels of the invention as well as vectors andconstructs that comprise such nucleic acid sequences. In someembodiments the invention includes expression of polypeptides encoded bythe nucleic acid sequences, in cells, tissues, and organisms.

Not all channelrhodopsins can be expressed in cells and utilized in thisfashion, because many do not traffic properly and/or function inmammalian cells. Many channelrhodopsins were screened in order toidentify ChR87, Chrimson, and Chronos as functioning better in mammaliancells than other classes of channelrhodopsins. In addition Chrimsonresponds strongly to far red light, and therefore, because otherchannelrhodopsins that depolarize cells respond strongly to ultravioletor blue light, Chrimson can be expressed in a separate population ofcells from a population of cells expressing one of these other opsins,allowing multiple colors of light to be used to excite these twopopulations of cells or neuronal projections from one site, at differenttimes.

In some embodiments of the invention, light-activated channels are usedto modify the transmembrane potential (and/or ionic composition) ofcells (and/or their sub-cellular regions, and their local environment).In particular, the use of inwardly rectifying cationic channels willdepolarize cells by moving positively charged ions from theextracellular environment to the cytoplasm. Under certain conditions,their use can decrease the intracellular pH (and/or cationconcentration) or increase the extracellular pH (and/or cationconcentration).

Compared to natural gene sequences conventionally used to depolarizeneurons, Chronos has demonstrably improved photocurrent generation atall illumination wavelengths except for red wavelength. In additionChronos can depolarize cells in response to <5 ms pulse of 50-100 uWmm⁻² of blue or green light with sufficient spectral independence frommost green or red channelrhodopsins such as ChR87 or Chrimson, thuspermitting multiple colors of light to be used to depolarize differentsets of cells in the same tissue, simply by expressing pumps withdifferent activation spectra genetically in different cells, and thenilluminating the tissue with different colors of light. In anon-limiting example of an embodiment, one set of cells in a tissue, forexample excitatory neurons, express Chrimson, and a second set expressChronos, illuminating the tissue with 630 nm light will preferentiallydepolarize the first set, and illuminating the tissue with 470 nm lightat low powers (<5 mW mm⁻²) will preferentially depolarize the secondset. Other pairs of targets that could be modulated with two colors oflight in the same illumination area include, but are not limited to twoprojections to/from one site, or combinations of the cell, itsprojections, and its organelles, given the ability to target themolecule sub-cellularly.

It has been identified that light-activated ion channels of theinvention are, in some embodiments of the invention, activated atdifferent wavelengths than previously identified light-activated ionchannels. Thus, light-activated ion channels of the invention can beused in either alone, using a selective light spectrum for activationand depolarization and can also be used in combination with otherlight-activated ion channels that utilize different wavelength of lightfor activation and depolarization, thus allowing two, three, four, ormore different wavelengths of light to be used to depolarize differentsets or subpopulations of cells in a tissue or organism by expressingchannels with different activation spectra in different cells and thenilluminating the tissue and/or organism with the appropriate wavelengthsof light to activate the channels and depolarize the cells.

According to some aspects of the invention, a light-activated ionchannel from Chlamydomonas noctigama or a derivative thereof may be usedin conjunction with a light-activated ion channel from Stigeocloniumhelveticum or a derivative thereof. The two light-activated ion channelsare sensitive to and can be activated with different wavelengths oflight than each other. As described herein, certain light-activated ionchannels of the invention can depolarize cells in strong response tolight with sufficient spectral independence from that of otherlight-activate ion channels of the invention, thus allowing multiplecolors of light to be used to depolarize different sets of cells in thesame tissue, by expressing channels with different activation spectragenetically in different cells, and then illuminating the tissue withdifferent colors of light in suitable dose to activate one type oflight-activated ion channel but not the other type of light-activatedion channel. In a non-limiting example, if a first subset of cells in atissue (e.g., excitatory neurons) express ChR88, and a second subsetexpress ChR90 light-activated ion channels of the invention, thenilluminating the tissue with a dose of light such as 625 nm, 2 mW/mm²will preferentially depolarize/drive spike in the first subset (ChR88),and illuminating the tissue with a dose of light such as 470 nm 0.2mW/mm² light will preferentially depolarize/drive spike in the secondsubset (ChR90). Other pairs of targets that could be modulated with twocolors of light in the same illumination area include, but are notlimited to two projections to/from one site, or combinations of thecell, its projections, and its organelles, given the ability to targetthe molecule sub-cellularly.

Taxonomy and Sequence Sources

In particular, the present invention includes, in part, novellight-activated ion channels and their use to depolarize cells. In somenon-limiting embodiments of the invention one or more newly identifiedlight-activated ion channels may be expressed in cells.

Some light-activated ion channels of the invention have amino acidsequences derived from Chlamydomonas rhodopsins that are naturallyexpressed in the genus Chlamydomonas noctigama, or another member of theChlamydomonadaceae family. Chlamydomonas noctigama are phytoplankton andcan be found in fresh water environments. Some embodiments of theinvention include isolated wild-type or modified nucleic acid and/oramino acid channelrhodopsin sequences from a member of thechlamydomonadaceae family, for example, from Chlamydomonas noctigama,and in some aspects, the invention also includes methods for their use.

Some light-activated ion channels of the invention have amino acidsequences derived from Stigeoclonium rhodopsins that are naturallyexpressed in the genus Stigeoclonium helveticum, or another member ofthe Chaetophoracea family. Stigeoclonium helveticum are green algae andcan be found in fresh water environments. Some embodiments of theinvention include isolated wild-type or modified nucleic acid and/oramino acid channelrhodopsin sequences from a member of theChaetophoracea family, for example, from Stigeoclonium helveticum, andin some aspects, the invention also includes methods for their use.

Some light-activated ion channels of the invention have amino acidsequences derived from Chloromonas rhodopsins that are naturallyexpressed in the genus Chloromonas subdivisa, or another member of theChlamydomonadaceae family. Chloromonas subdivisa are phytoplankton andcan be found in fresh water environments. Some embodiments of theinvention include isolated wild-type or modified nucleic acid and/oramino acid channelrhodopsin sequences from a member of theChlamydomonadaceae family, for example, from Chloromonas subdivisa, andin some aspects, the invention also includes methods for their use.

Some light-activated ion channels of the invention have amino acidsequences derived from Neochlorosarcina and some light-activated ionchannels of the invention have amino acid sequences derived fromHeterochlamydomonas inaequalis rhodopsins. Some embodiments of theinvention include isolated wild-type or modified nucleic acid and/oramino acid channelrhodopsin sequences from Neochlorosarcina or fromHeterochlamydomonas inaequalis, and in some aspects, the invention alsoincludes methods for their use. Sequences of light-activated ionchannels of the invention, may be derived from a Chloromonas sequence, aChlamydomonas sequence, or a Stigeoclonium sequence and may include awild-type or modified channelrhodopsin sequence, also referred to as aderivative sequence.

A modified light-activated ion channel polypeptide of the invention(also referred to as a derivative of a light-activated ion channel)versus a naturally occurring light activated ion channel may be alteredby an substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids,by an insertion and/or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore amino acids at one or several positions. Both a wild-typelight-activated ion channel polypeptide and derivatives thereof may havean identity of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, with the sequence of thetransmembrane region of the wild-type light-activated ion channelpolypeptide, as long as they retain channel functionality. For ChR87,the amino acid sequence of the transmembrane region is set forth as SEQID NO:13, which includes amino acid residues 81-315 of SEQ ID NO:11. ForChR88, the amino acid sequence of the transmembrane region is set forthas SEQ ID NO:4, which includes amino acid residues 86-320 of SEQ IDNO:2. For ChR90, the amino acid sequence of the transmembrane region isset forth as SEQ ID NO: 9, which includes amino acid residues 61-295 ofSEQ ID NO:7. A derivative or modified light-activated ion channelpolypeptide of the invention may retain an identity of 20% or more ofthe transmembrane amino acid sequence from which it was derived.

In contrast, the level of identity between a derivative or modifiedlight-activated ion channel of the invention and the wild-type fromwhich it is derived may be more constrained to maintain the function ofthe light-activated ion channel. The amino acid sequence of anon-transmembrane region of a derived or modified light-activated ionchannel of the invention, may have at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identity to the amino acid sequence of thewild-type light-activated ion channel polypeptide from which they arederived.

Thus, for example, in some embodiments, a light-activated ion channel ofthe invention may be a derivative of ChR87 and have at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the non-transmembraneregions of the wild-type polypeptide sequence (SEQ ID NO:11) of thelight-activated ion channel polypeptide from which it is derived. Inanother non-limiting example, in some embodiments, a light-activated ionchannel of the invention may be a derivative of ChR90 and have at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to thenon-transmembrane regions of the wild-type polypeptide sequence (SEQ IDNO:7) of the light-activated ion channel polypeptide from which it isderived. Similarly, in another example, in some embodiments, alight-activated ion channel of the invention may be a derivative ofChR88 and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identity to the non-transmembrane regions of the wild-type polypeptidesequence (SEQ ID NO:2) of the light-activated ion channel polypeptidefrom which it is derived. In another non-limiting example of alight-activated ion channel derived from ChR88, a polypeptide thatincludes K176R substitution in the amino acid sequence of SEQ ID NO:2functions as a light-activated ion channel of the invention.

As used herein, the term “identity” refers to the degree of relatednessbetween two or more polypeptide sequences, which may be determined bythe match between the sequences.

The percentage is obtained as the percentage of identical amino acids intwo or more sequences taking account of gaps and other sequencefeatures. The identity between polypeptide sequences can be determinedby means of known procedures. Algorithms and programs are available androutinely used by those in the art to determine identity betweenpolypeptide sequences. Non-limiting examples of programs and algorithmsinclude BLASTP, BLASTN and FASTA (Altschul et al., NCB NLM NIH BethesdaMd. 20894; Altschul et al., 1990), Online BLAST programs from theNational Library of Medicine are available, for example, atblast.ncbi.nlm.nih.gov/Blast.cgi.

One skilled in the art will understand that light-activated ion channelsof the invention can be identified based on sequence similarity orhomology to a light-activated ion channel disclosed herein. It will beunderstood that additional light-activated ion channels may beidentified using sequence alignment with one of the light-activated ionchannels or derivatives thereof identified herein.

Based on the teaching provided herein regarding the Cholormonassubdivisa, Chlamydomonas noctigama, Stigeoclonium helveticumchannelrhodopsin sequences having light-activated ion channel functionand activity, additional rhodopsin sequences with sufficient amino acidsequence homology to a ChR87, ChR88, or ChR90, respectively, can beidentified. The presence of functionality, e.g., activation of a channelby contact with suitable light can be determined using methods describedherein, and function light-activated ion channels of the invention canbe used in methods described herein. It is understood that that thelevel of sequence identity with a light-activated ion channel of theinvention plus functionality with respect to activation by suitablelight contact can be characteristics used to identify additionallight-activated ion channels using standard procedures for sequencealignment, comparisons, and assays for ion channel activity.

Light-activated ion channels of the invention are transmembrane channelpolypeptides that use light energy to open, permitting ion conductancethrough their pore, thus altering the potential of the membrane in whichthey are expressed. A non-limiting example of an ion that can be movedthrough a pore of the invention includes a sodium ion, a potassium ion,a calcium ion, a proton, etc. Routine methods may be used to measuredifferent ion currents for light-activated ion channels of theinvention. Light-activated ion channels of the invention can beactivated by sustained light and/or by light pulses and by permittingion conductance upon activation, light-activated ion channels of theinvention can depolarize cells and alter the voltage in cells andorganelles in which they are expressed.

The wild-type and modified (derived) Cholormonas subdivisa,Chlamydomonas noctigama, Stigeoclonium helveticum rhodopsin nucleic acidand amino acid sequences used in aspects and methods of the inventionare “isolated” sequences. As used herein, the term “isolated” used inreference to a polynucleotide, nucleic acid sequence, or polypeptidesequence of a rhodopsin, means a polynucleotide, nucleic acid sequence,or polypeptide sequence that is separate from its native environment andpresent in sufficient quantity to permit its identification or use.Thus, an isolated polynucleotide, nucleic acid sequence, or polypeptidesequence of the invention is a polynucleotide, nucleic acid sequence, orpolypeptide sequence that is not part of, or included in its native,wild-type cell or organism. For example, a nucleic acid or polypeptidesequence may be naturally expressed in a cell or organism of a member ofthe Chloromonas genus, but when the sequence is not part of or includedin a Chloromonas cell or organism it is considered to be isolated.Similarly, a nucleic acid or polypeptide sequence may be naturallyexpressed in a cell or organism of a member of the Chlamydomonas genus,but the sequence is not part of or included in a Chlamydomonas cell ororganism, it is considered to be isolated. Similarly, a nucleic acid orpolypeptide sequence may be naturally expressed in a cell or organism ofa member of the Stigeoclonium genus, but the sequence is not part of orincluded in a Stigeoclonium cell or organism, it is considered to beisolated. Thus, a nucleic acid or polypeptide sequence of a Chloromonas,Chlamydomonas, Stigeoclonium, or other light-activated ion channelnucleic acid or polypeptide that is present in a vector, in aheterologous cell, tissue, or organism, etc., is still considered to bean isolated sequence. As used herein the term “host” used in referenceto a membrane or cell in which a light-activated ion channel of theinvention is expressed, means a membrane or cell that is not a cell ormembrane in which the light-activated ion channel is expressed innature. Thus a host membrane, cell, tissue, or organism for alight-activated ion channel molecule of the invention (such as alight-activated ion channel polypeptide or its encoding nucleic acid),as used herein is a membrane, cell, tissue, or organism in which thelight-activated ion channel molecule of the invention does not naturallyoccur and in which the light-activated ion channel is not naturallyexpressed. Examples of a host membrane, cell, tissue, or organisminclude, but are not limited to mammalian (including but not limited tonon-human primate, human, etc.), insect, and avian membranes, cells, andtissues; as well as organisms such as mammals, insects, and birds. Theterm “heterologous” as used herein, means a membrane, cell, tissue, ororganism that is not the native cell, tissue, or organism, and alight-activated ion channel polypeptide of the invention or its encodingnucleic acid may be present in a heterologous membrane, cell, tissue, ororganism. The terms, “protein”, “polypeptides”, and “peptides” are usedinterchangeably herein.

Light-Activated Ion Channel Sequences Including Modified and DerivedSequences

A light-activated ion channel of the invention may comprise a wild-typepolypeptide sequence or may be a modified polypeptide sequence. As usedherein the term “modified” or “modification” in reference to a nucleicacid or polypeptide sequence refers to a change of one, two, three,four, five, six, or more amino acids in the sequence as compared to thewild-type or other sequence from which it was derived. For example, amodified polypeptide sequence may be identical to a wild-typepolypeptide sequence except that it has one, two, three, four, five, ormore amino acid substitutions, deletions, insertions, or combinationsthereof. In some embodiments of the invention a modified sequence mayinclude one, two, three, four, or more amino acid substitutions in awild-type light-activated ion channel polypeptide sequence, or in anyother light-activated ion channel polypeptide sequence of the invention.

It will be understood that sequences of light-activated ion channels ofthe invention may be derived from various members of the Chloromonasgenus, Chlamydomonas genus, Stigeoclonium genus, Neochlorosarcina genus,or Heterochlamydomonas genus.

The invention, in some aspects also includes light-activated ion channelpolypeptides having one or more substitutions or other modificationsfrom those described herein. For example, sequences of light-activatedion channel polypeptides provided herein can be modified with one ormore substitutions, deletions, insertions, or other modifications andsuch derivative light-activated ion channels can be tested using methodsdescribed herein for characteristics including, but not limited to:expression, cell localization, activation and depolarization in responseto contact with light using methods disclosed herein. Exemplarymodifications include, but are not limited to conservative amino acidsubstitutions, which will produce molecules having functionalcharacteristics similar to those of the molecule from which suchmodifications are made. Conservative amino acid substitutions aresubstitutions that do not result in a significant change in the activityor tertiary structure of a selected polypeptide or protein. Suchsubstitutions typically involve replacing a selected amino acid residuewith a different residue having similar physico-chemical properties. Forexample, substitution of Glu for Asp is considered a conservativesubstitution because both are similarly-sized negatively-charged aminoacids. Groupings of amino acids by physico-chemical properties are knownto those of skill in the art. The following groups each contain aminoacids that are conservative substitutions for one another: 1) Alanine(A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine(Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). Light-activatedion channels that include modifications, including but not limited toone, two, three, four, or more conservative amino acid substitutions canbe identified and tested for characteristics including, but not limitedto: expression, cell localization, activation and depolarization anddepolarization-effects in response to contact with light using methodsdisclosed herein. As described elsewhere herein, in some polypeptideregions such as the transmembrane region of a light-activated ionchannel of the invention, may include modifications that result in lessthan 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% identitywith the sequence from which its derived, yet may have at least 90%,95%, 96%, 97%, 98%, 99%, or 100% identity in its non-transmembraneregions of the polypeptide.

Sequence identity can be determined using standard techniques known inthe art.

Light—activated ion channel polypeptides of the invention may be shorteror longer than the light-activated ion channel polypeptide sequences setforth herein. Thus, a light-activated ion channel polypeptide may be afull-length polypeptide or functional fragment thereof. In addition,nucleic acids of the invention may be used to obtain additional codingregions, and thus additional polypeptide sequences, using techniquesknown in the art.

In some aspects of the invention, substantially similar light-activatedion channel polypeptide sequences may have at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity to alight-activated ion channel polypeptide sequence disclosed herein,non-limiting examples of which include as ChR62, ChR93, ChR87, ChR88,ChR90, etc. Art-known alignment methods and tools can be used to alignsubstantially similar sequences permitting positional identification ofamino acids that may be modified as described herein to prepare alight-activated ion channel of the invention. Standard sequence analysistools and computer programs, such as those used for alignment, etc. canbe used to identify light-activated ion channels of the invention thatshare one or more functional properties with a light-activated ionchannel described herein.

Sequence modifications can be in one or more of three classes:substitutions, insertions, or deletions. These modified sequences,(which may also be referred to as variants, or derivatives) ordinarilyare prepared by site specific mutagenesis of nucleic acids in the DNAencoding a light-activated ion channel polypeptide, using cassette orPCR mutagenesis or other techniques known in the art, to produce DNAencoding the modified light-activated ion channel, and thereafterexpressing the DNA in recombinant cell culture. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the light-activated ion channels of theinvention. Modified light-activated ion channels generally exhibit thesame qualitative biological activity as the naturally occurringlight-activated ion channel (e.g., wild-type), although variants canalso be selected that have modified characteristics.

A site or region for introducing an amino acid sequence modification maybe predetermined, and the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed modified light-activated ion channel screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis.

Amino acid substitutions are typically of single residues; andinsertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions may range from about 1 to about 20 residues, although in somecases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final modified light-activated ion channel of theinvention. Generally these changes are done on a few amino acids tominimize the alteration of the molecule. However, larger changes may betolerated in certain circumstances.

Variants of light-activated ion channels set forth herein, may exhibitthe same qualitative light-activated ion channel activity as one or moreof the sequences set forth herein, such as ChR62, ChR93, ChR87, ChR88,or ChR90, but may show some altered characteristics such as alteredphotocurrent, stability, speed, compatibility, and toxicity, or acombination thereof. For example, the polypeptide can be modified suchthat it has an increased photocurrent and/or has less toxicity thananother light-activated ion channel polypeptide.

A modified (or derived) light-activated ion channel polypeptide of theinvention can incorporate unnatural amino acids as well as natural aminoacids. An unnatural amino acid can be included in a light-activated ionchannel of the invention to enhance a characteristic such asphotocurrent, stability, speed, compatibility, or to lower toxicity,etc.

According to principles of this invention, the performance oflight-activated ion channel molecules or classes of molecules can betuned for optimal use, including in the context of their use inconjunction with other molecules or optical apparatus. For example, inorder to achieve optimal contrast for multiple-color stimulation, onemay desire to either improve or decrease the performance of one moleculewith respect to one another, by the appendage of trafficking enhancingsequences or creation of genetic variants by site-directed mutagenesis,directed evolution, gene shuffling, or altering codon usage.light-activated ion channel molecules or classes of molecules may haveinherently varying spectral sensitivity. This may be used to advantagein vivo (where scattering and absorption will vary with respect towavelength, coherence, and polarization), by tuning the linearity ornon-linearity of response to optical illumination with respect to time,power, and illumination history.

In some embodiments, the invention includes the use of targetedsite-directed mutagenesis at specific amino acid residues ofchannelrhodopsins including but not limited to residues ofchannelrhodopsins of the Chloromonas genus, Chlamydomonas genus,Stigeoclonium genus, Neochlorosarcina genus, or Heterochlamydomonasgenus. Specific locations for single mutations can be identified andalone, or in combination with two or more additional mutations can beplaced into a channelrhodopsin sequence and tested with respect to theiractivation and photocurrent amplitude. Thus, sequences oflight-activated ion channels of the invention, and/or similarchannelrhodopsin sequences can be modified and the resultingpolypeptides tested using methods disclosed herein.

Another aspect of the invention provides nucleic acid sequences thatcode for a light-activated ion channel of the invention. It would beunderstood by a person of skill in the art that light-activated ionchannel polypeptides of the present invention can be coded for byvarious nucleic acids. Each amino acid in the protein is represented byone or more sets of 3 nucleic acids (codons). Because many amino acidsare represented by more than one codon, there is not a unique nucleicacid sequence that codes for a given protein. It is well understood bythose of skill in the art how to make a nucleic acid that can code forlight-activated ion channel polypeptides of the invention by knowing theamino acid sequence of the protein. A nucleic acid sequence that codesfor a polypeptide or protein is the “gene” of that polypeptide orprotein. A gene can be RNA, DNA, or other nucleic acid than will codefor the polypeptide or protein.

It is understood in the art that the codon systems in differentorganisms can be slightly different, and that therefore where theexpression of a given protein from a given organism is desired, thenucleic acid sequence can be modified for expression within thatorganism. Thus, in some embodiments, a light-activated ion channelpolypeptide of the invention is encoded by a mammalian-codon-optimizednucleic acid sequence, which may in some embodiments be a human-codonoptimized nucleic acid sequence. An aspect of the invention provides anucleic acid sequence that codes for a light-activated ion channel thatis optimized for expression with a mammalian cell. In some embodimentsof the invention, a nucleic acid that encodes a light-activated ionchannel of the invention includes a nucleic acid sequence optimized forexpression in a human cell.

Delivery of Light-Activated Ion Channels

Delivery of a light-activated ion channel polypeptide to a cell and/orexpression of a light-activated ion channel in a cell can be done usingart-known delivery means.

In some embodiments of the invention a light-activated ion channelpolypeptide of the invention is included in a fusion protein. It is wellknown in the art how to prepare and utilize fusion proteins thatcomprise a polypeptide sequence. In certain embodiments of theinvention, a fusion protein can be used to deliver a light-activated ionchannel to a cell and can also in some embodiments be used to target alight-activated ion channel of the invention to specific cells or tospecific cells, tissues, or regions in a subject. Targeting and suitabletargeting sequences for deliver to a desired cell, tissue or region canbe performed using art-known procedures.

It is an aspect of the invention to provide a light-activated ionchannel polypeptide of the invention that is non-toxic, or substantiallynon-toxic in cells in which it is expressed. In the absence of light, alight-activated ion channel of the invention does not significantlyalter cell health or ongoing electrical activity in the cell in which itis expressed.

In some embodiments of the invention, a light-activated ion channel ofthe invention is genetically introduced into a cellular membrane, andreagents and methods are provided for genetically targeted expression oflight-activated ion channel polypeptides, including ChR87, ChR88, ChR90,ChR62, ChR93, ChR88 K176R, or a derivative thereof, etc. Genetictargeting can be used to deliver light-activated ion channelpolypeptides to specific cell types, to specific cell subtypes, tospecific spatial regions within an organism, and to sub-cellular regionswithin a cell. Genetic targeting also relates to the control of theamount of light-activated ion channel polypeptide expressed, and thetiming of the expression.

Some embodiments of the invention include a reagent for geneticallytargeted expression of a light-activated ion channel polypeptide,wherein the reagent comprises a vector that contains the gene for thelight-activated ion channel polypeptide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. The term “vector”also refers to a virus or organism that is capable of transporting thenucleic acid molecule. One type of vector is an episome, i.e., a nucleicacid molecule capable of extra-chromosomal replication. Some usefulvectors are those capable of autonomous replication and/or expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. Other useful vectors, include, but arenot limited to viruses such as lentiviruses, retroviruses, adenoviruses,and phages. Vectors useful in some methods of the invention cangenetically insert light-activated ion channel polypeptides intodividing and non-dividing cells and can insert light-activated ionchannel polypeptides to cells that are in vivo, in vitro, or ex vivocells.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. Non-limitingexamples of suitable expression vectors and methods for their use areprovided herein.

In certain embodiments of the invention, a vector may be a lentiviruscomprising the gene for a light-activated ion channel of the invention,such as ChR87, ChR88, ChR90, ChR62, ChR93, ChR88 K176R, or a derivativeor variant thereof. A lentivirus is a non-limiting example of a vectorthat may be used to create stable cell line. The term “cell line” asused herein is an established cell culture that will continue toproliferate given the appropriate medium.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of a light-activated ionchannel polypeptide in a wide variety of cell types—thus a promoter fora gene that is widely expressed in a variety of cell types, for examplea “housekeeping gene” can be used to express a light-activated ionchannel polypeptide in a variety of cell types. Non-limiting examples ofgeneral promoters are provided elsewhere herein and suitable alternativepromoters are well known in the art.

In certain embodiments of the invention, a promoter may be an induciblepromoter, examples of which include, but are not limited totetracycline-on or tetracycline-off, or tamoxifen-inducible Cre-ER.

Methods of Use of Light Activated Ion Channels of the Invention

Light activated ion channels of the invention are well suited fortargeting cells and specifically altering voltage-associated cellactivities. In some embodiments of the invention, light-activated ionchannels of the invention can utilized to introduce cations into cells,thus activating endogenous signaling pathways (such as calcium dependentsignaling), and then drugs are applied that modulate the response of thecell (using a calcium or voltage-sensitive dye). This allows new kindsof drug screening using just light to activate the channels of interest,and using just light to read out the effects of a drug on the channelsof interest.

Chrimson is far-red-activatable, and thus allows excitation of cellswith a color of light heretofore not used in biotechnology forexcitation of cells. By using for example, Chrimson and Chronostogether, excitation of two different populations of cells in the sametissue or in the same culture dish becomes possible. This simultaneous,two-color excitation is particularly promising for complex tissues suchas the brain.

The performance of the above said molecules or classes of molecules canbe tuned for optimal use, particularly in context of their use inconjunction with other molecules or optical apparatus. Such tuning canbe done using standard methods known in the art. For example, in orderto achieve optimal contrast for multiple-color stimulation, one maydesire to either improve or decrease the performance of one moleculewith respect to one another, by the appendage of trafficking enhancingsequences or creation of genetic variants by site-directed mutagenesis,directed evolution, gene shuffling, or altering codon usage. Moleculesor classes of molecules may have inherently varying spectral sensitivitythat may be functionally advantageous in vivo (where scattering andabsorption will vary with respect to wavelength, coherence, andpolarization), by tuning the linearity or non-linearity of response tooptical illumination with respect to time, power, and illuminationhistory.

According to certain principles of this invention, cations may beintroduced into cells, thus activating endogenous signaling pathways(such as calcium dependent signaling), and drugs may be applied thatmodulate the response of the cell (using a calcium or voltage-sensitivedye). This enables new kinds of drug screening using just light toactivate the channels of interest, and using just light to read out theeffects of a drug on the channels of interest.

Another aspect of the invention is the use of light-activated channel todecrease the pH of the cell. Such a technique may be used to treatalkalosis.

Another aspect of the invention may involve the use of light-activatedproton pumps for the coupled effect of hyperpolarization andintracellular alkalinization. For example, both phenomena can inducespontaneous spiking in neurons by triggering hyperpolarization-inducedcation currents or pH-dependent hyper-excitability.

Another aspect of the invention is to generate sub-cellular voltage orpH gradients, particularly at synapses and in synaptic vesicles to altersynaptic transmission, and mitochondria to improve ATP synthesis.

Another aspect of the invention is the use of far-red (660 nm) light toperform non-invasive transcranial and/or transdural stimulation tomodulate neural circuits.

Another aspect of the invention is the various compositions of matterthat have now been reduced to practice, for example: plasmids encodingfor the above genes have been prepared; lentiviruses carrying payloadsencoding for the above genes have been prepared; adeno-associatedviruses carrying payloads encoding for the above genes have beenprepared; cells expressing the above genes have been prepared; andanimals expressing those genes have been prepared. (See for example: USPatent Publication 20110165681, incorporated herein by reference in itsentirety).

Working operation of a prototype of this invention was demonstrated bygenetically expressing light-activated ion channel molecules of theinvention in excitable cells, illuminating the cells with suitablewavelengths of light, and demonstrating rapid depolarization of thecells in response to the light, as well as rapid release fromdepolarization upon cessation of light. Depending on the particularimplementation, methods of the invention allow light control of cellularfunctions in vivo, ex vivo, and in vitro.

In non-limiting examples of methods of the invention, channelrhodopsinsof the invention and derivatives thereof are used in mammalian cellswithout need for any kind of chemical supplement, and in normal cellularenvironmental conditions and ionic concentrations. For example, genesencoding channelrhodopsins of Chlamydomonas and Stigeoclonium have beenused in exemplary implementations of the invention. These sequences inhumanized or mouse-optimized form allow depolarization at wavelengthsdescribed herein.

As used herein, the term “ion channel” means a transmembrane polypeptidethat forms a pore, which when activated opens, permitting ionconductance through the pore across the membrane. Many ion channels donot express well in a cell and/or their expression may be toxic to thecell and reduce cell health. Thus it was necessary to prepare and screennumerous channelrhodopsin light-activated ion channel polypeptides toidentify light-activated ion channels of the invention that can beexpressed in cells without significantly reducing cell health andviability.

Light-activated ion channels of the invention have been found to besuitable for expression and use in mammalian cells without need for anykind of chemical supplement, and in normal cellular environmentalconditions and ionic concentrations. Light-activated ion channels of theinvention have been found to differ from previously identified channelsin that the Chronos light-activated ion channels activate at wavelengthsof light ranging from 365 nm to 630 nm, with an optimal activation fromlight ranging from 430 nm to 550 nm, and a peak optimal activation at awavelength of 500 nm. Chrimson light-activated ion channels activate atwavelengths of light in a range of 365 nm to 700 nm, with an optimalactivation from light ranging from 530 nm to 640 nm, and a peak optimalactivation at a wavelength of 590 nm.

Cells and Subjects

A cell used in methods and with sequences of the invention may be anexcitable cell or a non-excitable cell. A cell in which alight-activated ion channel of the invention may be expressed and may beused in methods of the invention include prokaryotic and eukaryoticcells. Useful cells include but are not limited to mammalian cells.Examples of cells in which a light-activated ion channel of theinvention may be expressed are excitable cells, which include cells ableto produce and respond to electrical signals. Examples of excitable celltypes include, but are not limited to neurons, muscles, cardiac cells,and secretory cells (such as pancreatic cells, adrenal medulla cells,pituitary cells, etc.).

Non-limiting examples of cells that may be used in methods of theinvention include: neuronal cells, nervous system cells, cardiac cells,circulatory system cells, visual system cells, auditory system cells,secretory cells, endocrine cells, or muscle cells. In some embodiments,a cell used in conjunction with the invention may be a healthy normalcell, which is not known to have a disease, disorder or abnormalcondition. In some embodiments, a cell used in conjunction with methodsand channels of the invention may be an abnormal cell, for example, acell that has been diagnosed as having a disorder, disease, orcondition, including, but not limited to a degenerative cell, aneurological disease-bearing cell, a cell model of a disease orcondition, an injured cell, etc. In some embodiments of the invention, acell may be a control cell.

Light-activated ion channels of the invention may be expressed in cellsfrom culture, cells in solution, cells obtained from subjects, and/orcells in a subject (in vivo cells). Light-activated ion channels may beexpressed and activated in cultured cells, cultured tissues (e.g., brainslice preparations, etc.), and in living subjects, etc. As used herein,a the term “subject” may refer to a human, non-human primate, cow,horse, pig, sheep, goat, dog, cat, rodent, fly or any other vertebrateor invertebrate organism.

Controls and Candidate Compound Testing

Light-activated ion channels of the invention and methods usinglight-activated ion channels of the invention can be utilized to assesschanges in cells, tissues, and subjects in which they are expressed.Some embodiments of the invention include use of light-activated ionchannels of the invention to identify effects of candidate compounds oncells, tissues, and subjects. Results of testing a light-activated ionchannel of the invention can be advantageously compared to a control. Insome embodiments of the invention one or more light-activated ionchannels of the invention, non-limiting examples of which are ChR87,ChR88, ChR90, ChR93, ChR62, ChR88 K176R, or a derivative thereof, may beexpressed in a cell population and used to test the effect of candidatecompounds on the cells.

As used herein a control may be a predetermined value, which can take avariety of forms. It can be a single cut-off value, such as a median ormean. It can be established based upon comparative groups, such as cellsor tissues that include the light-activated ion channel and arecontacted with light, but are not contacted with the candidate compoundand the same type of cells or tissues that under the same testingcondition are contacted with the candidate compound. Another example ofcomparative groups may include cells or tissues that have a disorder orcondition and groups without the disorder or condition. Anothercomparative group may be cells from a group with a family history of adisease or condition and cells from a group without such a familyhistory. A predetermined value can be arranged, for example, where atested population is divided equally (or unequally) into groups based onresults of testing. Those skilled in the art are able to selectappropriate control groups and values for use in comparative methods ofthe invention.

As a non-limiting example of use of a light-activated ion channel toidentify a candidate therapeutic agent or compound, a light-activatedion channel of the invention may be expressed in an excitable cell inculture or in a subject and the excitable cell may be contacted with alight that activates the light-activated ion channel and with acandidate therapeutic compound. In one embodiment, a test cell thatincludes a light-activated ion channel of the invention can be contactedwith a light that depolarizes the cell and also contacted with acandidate compound. The cell, tissue, and/or subject that include thecell can be monitored for the presence or absence of a change thatoccurs in the test conditions versus the control conditions. Forexample, in a cell, a change may be a change in the depolarization or ina depolarization-mediated cell characteristic in the test cell versus acontrol cell, and a change in depolarization or thedepolarization-mediated cell characteristic in the test cell compared tothe control may indicate that the candidate compound has an effect onthe test cell or tissue that includes the cell. In some embodiments ofthe invention, a depolarization-mediated cell characteristic may be a anaction potential, pH change in a cell, release of a neurotransmitter,etc. and may in come embodiments, include a downstream effect on one ormore additional cells, which occurs due to the depolarization of thecell that includes the light-activated ion channel. Art-known methodscan be sued to assess depolarization and depolarization-mediated cellcharacteristics and changes to the depolarization ordepolarization-mediated cell characteristics upon activation of alight-activated ion channel of the invention, with or without additionalcontact with a candidate compound.

Candidate-compound identification methods of the invention that areperformed in a subject, may include expressing a light-activated ionchannel in a subject, contacting the subject with a light under suitableconditions to activate the light-activated ion channel and depolarizethe cell, and administering to the subject a candidate compound. Thesubject is then monitored to determine whether any change occurs thatdiffers from a control effect in a subject. Thus, for example, a cell inculture can be contacted with a light appropriate to activate alight-activated ion channel of the invention in the presence of acandidate compound. A result of such contact with the candidate compoundcan be measured and compared to a control value as a determination ofthe presence or absence of an effect of the candidate compound.

Methods of identifying effects of candidate compounds usinglight-activated ion channels of the invention may also includeadditional steps and assays to further characterizing an identifiedchange in the cell, tissue, or subject when the cell is contacted withthe candidate compound. In some embodiments, testing in a cell, tissue,or subject can also include one or more cells that has a light-activatedion channel of the invention, and that also has one, two, three, or moreadditional different light-activated ion channels, wherein at least one,two, three, four, or more of the additional light-activated ion channelsis activated by contact with light having a different wavelength thanused to activate the Chronos, Chrimson, ChR87, or derivative thereof,light-activated ion channel of the invention. In a non-limiting exampleof a candidate drug identification method of the invention, cells thatinclude a light-activated ion channel of the invention are depolarized,thus triggering release of a neurotransmitter from the cell, and thendrugs are applied that modulate the response of the cell todepolarization (determined for example using patch clamping methods orother suitable art-known means). Such methods enable new kinds of drugscreening using just light to activate the channels of interest, andusing just light to read out the effects of a drug on the channels andchannel-containing cells of interest.

In some embodiments, light-activated ion channel polypeptides of theinvention can be used in test systems and assays for assessing membraneprotein trafficking and physiological function in heterologouslyexpressed systems and the use of use of light-activated channels todepolarize a cell.

In some aspects of the invention, two-color assays can be performed. Forexample, Chronos (for blue light activation) and Chrimson (for red lightactivation) can be expressed in separate sets cells that representnon-overlapping neuronal populations. Following expression, the cellpopulation can be exposed to light and the wavelength and timing and“dose” of light can be optimized. As used herein the term “dose” inreference to light, may take into account of wavelength, pulse length,intensity, of the light with which a light-activated ion channel of theinvention is contacted.

A non-limiting example of a procedure for optimizing the use oftwo-color activated populations of cells is provided as follows. Apopulation that has Chronos and Chrimson expressed in differentsub-populations is contacted with blue light having a wavelength between400 nm and 500 nm, or between 450 nm to 500 nm, and having a pulse widthof between 1 and 5 ms for activation. A pulse width of 5 ms provides forminimum sub-threshold crosstalk in the blue light, which is defined as<15 mV, <10 mV, and optimally as <5 mV. The maximum blue light powerthat can be used is determined using by patch clamping Chrimsonexpressing cells, illuminating with blue light and measuring voltagedeflection. Optimally using blue light power such that maximum voltagedeflection is <10 mV, which in some embodiments may be 0.4 to 0.6mW/mm². The optimal blue light power that can be used to drive Chronosis determined using the same conditions as above, except using lowerlight power, such as 50 μW/mm² to 0.4 mW/mm², which in some embodimentsmay be 0.2 mW/mm². Power depends on expression system and cell type usedto prepare the population. The population can be contacted with redlight having a wavelength between 600 nm and 700 nm, or 620 nm and 640nm, and with a pulse width of between 1 and 5 ms for activation, whichin some embodiments may be optimized at 5 ms. In certain embodiments ofthe invention, the optimal light power to drive Chrimson in the red maybe determined by ramping light powers from for example, 0.1 mW/mm² to100 mW/mm², or from 0.5 mW/mm² to 10 mW/mm². The method may be optimizedsuch that a minimum red light power is used to achieve 100% spiking forChrimson.

It will be understood that other sets of 2, 3, 4, or morelight-activated ion channels may be expressed in separate subpopulationsof a population of cells and then exposed to doses of light in a manneras described here to optimize their use in assays and treatments of theinvention. A non-limiting example of a process to prepare and use amulti-light activated population of cells is as follows. A firstlight-activated ion channel is expressed in a first subpopulation of apopulation of cells; a second light-activated ion channel is expressedin a second subpopulation of the population of cells, wherein the firstand second subpopulations are non-overlapping subpopulations, and thefirst light-activated ion channel and second light activated ion channelare have ranges of activating light wavelengths that do not entirelyoverlap. The population of cells is contacted with a plurality of firstlight test doses comprising combinations of wavelength, pulse width, andpower that activate the first subpopulation, and the transmembranevoltage deflection is measured in a cell of the second subpopulation ofcells contacted with the first light test doses. The first light testdose that includes a maximum light power that activates the lightactivated ion channel in first to subpopulation of cells and results ina minimum sub-threshold transmembrane voltage deflection in the secondsubpopulation of cells is determined. The population of cells is thencontacted with a plurality of first light test doses comprising a lowerpower than the maximum first light power that was determined, and afirst light test doses that activate the first light activated ionchannel (at the lower powers) are determined. The population of cells isthen contacted with a plurality of second light test doses that includecombinations of light wavelength, pulse width, and power that activatethe second subpopulation, and a second light test dose comprising asecond light power that activates the second subpopulation of cells isdetermined. Assays can be performed using such a population of cells,that includes contacting the population of cells with the first lighttest dose and the second light test dose determined using the stepsabove. The above-described process of optimizing light dose parametersfor multi-light activated ion channels can be used to design andimplement assays that include light-activated ion channels of theinvention, as well as other light-activated ion channels that are knownin the art.

Methods of Treating

Some aspects of the invention include methods of treating a disorder orcondition in a cell, tissue, or subject using light-activated ionchannels of the invention. Treatment methods of the invention mayinclude administering to a subject in need of such treatment, atherapeutically effective amount of a light-activated ion channel of theinvention to treat the disorder. It will be understood that a treatmentmay be a prophylactic treatment or may be a treatment administeredfollowing the diagnosis of a disease or condition. A treatment of theinvention may reduce or eliminate a symptom or characteristic of adisorder, disease, or condition or may eliminate the disorder, disease,or condition itself. It will be understood that a treatment of theinvention may reduce or eliminate progression of a disease, disorder orcondition and may in some instances result in the regression of thedisease, disorder, or condition. A treatment need to entirely eliminatethe disease, disorder, or condition to be effective. In some embodimentsof the invention one or more light-activated ion channels of theinvention, non-limiting examples of which are ChR87, ChR88, ChR90,ChR93, ChR62 may be expressed in a cell population and used in methodsto treat a disorder or condition.

Administration of a light-activated ion channel of the invention mayinclude administration pharmaceutical composition that includes a cell,wherein the cell expresses the light-activated ion channel.Administration of a light-activated ion channel of the invention mayinclude administration of a pharmaceutical composition that includes avector, wherein the vector comprises a nucleic acid sequence encodingthe light-activated ion channel and the administration of the vectorresults in expression of the light-activated ion channel in a cell inthe subject.

An effective amount of a light-activated ion channel is an amount thatincreases the level of the light-activated ion channel in a cell, tissueor subject to a level that is beneficial for the subject. An effectiveamount may also be determined by assessing physiological effects ofadministration on a cell or subject, such as a decrease in symptomsfollowing administration. Other assays will be known to one of ordinaryskill in the art and can be employed for measuring the level of theresponse to a treatment. The amount of a treatment may be varied forexample by increasing or decreasing the amount of the light-activatedion channel administered, by changing the therapeutic composition inwhich the light-activated ion channel is administered, by changing theroute of administration, by changing the dosage timing, by changing theactivation amounts and parameters of a light-activated ion channel ofthe invention, and so on. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated; the severity of the condition, the durationof the treatment, the nature of the concurrent therapy (if any), thespecific route of administration, and the like factors within theknowledge and expertise of the health practitioner. For example, aneffective amount may depend upon the location and number of cells in thesubject in which the light-activated ion channel is to be expressed. Aneffective amount may also depend on the location of the tissue to betreated.

Effective amounts will also depend, of course, on the particularcondition being treated, the severity of the condition, the individualpatient parameters including age, physical condition, size and weight,the duration of the treatment, the nature of concurrent therapy (ifany), the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of a composition to increase the level of alight-activated ion channel, and/or to alter the length or timing ofactivation of a light-activated ion channel in a subject (alone or incombination with other therapeutic agents) be used, that is, the highestsafe dose or amount according to sound medical judgment. It will beunderstood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

A light-activated ion channel of the invention (for example, ChR87,ChR88, ChR90, ChR93, ChR62, or ChR88 K176R, or a derivative thereof) maybe administered using art-known methods. In some embodiments a nucleicacid that encodes a light-activated ion channel polypeptide of theinvention is administered to a subject and in certain embodiments alight-activated ion channel polypeptide is administered to a subject.The manner and dosage administered may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. The absolute amount administered will depend upon avariety of factors, including the material selected for administration,whether the administration is in single or multiple doses, andindividual subject parameters including age, physical condition, size,weight, and the stage of the disease or condition. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation.

Pharmaceutical compositions that deliver light-activated ion channels ofthe invention may be administered alone, in combination with each other,and/or in combination with other drug therapies, or other treatmentregimens that are administered to subjects. A pharmaceutical compositionused in the foregoing methods preferably contain an effective amount ofa therapeutic compound that will increase the level of a light-activatedion channel polypeptide to a level that produces the desired response ina unit of weight or volume suitable for administration to a subject.

The dose of a pharmaceutical composition that is administered to asubject to increase the level of light-activated ion channel in cells ofthe subject can be chosen in accordance with different parameters, inparticular in accordance with the mode of administration used and thestate of the subject. Other factors include the desired period oftreatment. In the event that a response in a subject is insufficient atthe initial doses applied, higher doses (or effectively higher doses bya different, more localized delivery route) may be employed to theextent that patient tolerance permits. The amount and timing ofactivation of a light-activated ion channel of the invention (e.g.,light wavelength, length of light contact, etc.) that has beenadministered to a subject can also be adjusted based on efficacy of thetreatment in a particular subject. Parameters for illumination andactivation of light-activated ion channels that have been administeredto a subject can be determined using art-known methods and withoutrequiring undue experimentation.

Various modes of administration will be known to one of ordinary skillin the art that can be used to effectively deliver a pharmaceuticalcomposition to increase the level of a light-activated ion channel ofthe invention in a desired cell, tissue or body region of a subject.Methods for administering such a composition or other pharmaceuticalcompound of the invention may be topical, intravenous, oral,intracavity, intrathecal, intrasynovial, buccal, sublingual, intranasal,transdermal, intravitreal, subcutaneous, intramuscular and intradermaladministration. The invention is not limited by the particular modes ofadministration disclosed herein. Standard references in the art (e.g.,Remington's Pharmaceutical Sciences, 18th edition, 1990) provide modesof administration and formulations for delivery of variouspharmaceutical preparations and formulations in pharmaceutical carriers.Other protocols which are useful for the administration of a therapeuticcompound of the invention will be known to one of ordinary skill in theart, in which the dose amount, schedule of administration, sites ofadministration, mode of administration (e.g., intra-organ) and the likevary from those presented herein.

Administration of a cell or vector to increase light-activated ionchannel levels in a mammal other than a human; and administration anduse of light-activated ion channels of the invention, e.g. for testingpurposes or veterinary therapeutic purposes, is carried out undersubstantially the same conditions as described above. It will beunderstood by one of ordinary skill in the art that this invention isapplicable to both human and animals. Thus this invention is intended tobe used in husbandry and veterinary medicine as well as in humantherapeutics.

In some aspects of the invention, methods of treatment using alight-activated ion channel of the invention are applied to cellsincluding but not limited to a neuronal cell, a nervous system cell, aneuron, a cardiac cell, a circulatory system cell, a visual system cell,an auditory system cell, a muscle cell, or an endocrine cell, etc.Disorders and conditions that may be treated using methods of theinvention include, injury, brain damage, degenerative neurologicalconditions (e.g., Parkinson's disease, Alzheimer's disease, seizure,vision loss, hearing loss, etc.

Disorders, Diseases and Conditions

Light-activated ion channels of the invention may be used to targetcells and membranes, and to alter voltage-associated cell activities. Insome aspects of the invention, a light-activated ion channel of theinvention may be used to decrease the pH of a cell in which it isexpressed. Such a technique may be used to treat alkalosis.

Another aspect of the invention includes methods of usinglight-activated proton pumps in conjunction with the use oflight-activated ion channels of the invention for the coupled effect ofhyperpolarization and intracellular alkalinization. For example, bothphenomena can induce spontaneous spiking in neurons by triggeringhyperpolarization-induced cation currents or pH-dependenthyper-excitability.

Another aspect of the invention is to express light-activated ionchannels of the invention into cell membranes and then to activate thelight-activated ion channels and generate sub-cellular voltage or pHgradients, particularly at synapses and in synaptic vesicles to altersynaptic transmission, and mitochondria to improve ATP synthesis.

In some embodiments, methods and light-activated ion channels of theinvention may be used for the treatment of visual system disorders, forexample to treat vision reduction or loss. A light-activated ion channelof the invention may be administered to a subject who has a visionreduction or loss and the expressed light-activated ion channel canfunction as light-sensitive cells in the visual system, therebypermitting a gain of visual function in the subject.

The present invention in some aspects, includes preparing nucleic acidsequences and polynucleotide sequences; expressing in cells andmembranes polypeptides encoded by the prepared nucleic acid andpolynucleotide sequences; illuminating the cells and/or membranes withsuitable light, and demonstrating rapid depolarization of the cellsand/or a change in conductance across the membrane in response to light,as well as rapid release from depolarization upon cessation of light.The ability to controllably alter voltage across membranes and celldepolarization with light has been demonstrated. The present inventionenables light-control of cellular functions in vivo, ex vivo, and invitro, and the light activated ion channels of the invention and theiruse, have broad-ranging applications for drug screening, treatments, andresearch applications, some of which are describe herein.

In illustrative implementations of this invention, the ability tooptically perturb, modify, or control cellular function offers manyadvantages over physical manipulation mechanisms. These advantagescomprise speed, non-invasiveness, and the ability to easily span vastspatial scales from the nanoscale to macroscale.

The reagents use in the present invention (and the class of moleculesthat they represent), allow, at least: currents activated by lightwavelengths not useful in previous light-activated ion channels, lightactivated ion channels that when activated, permit effectively zerocalcium conductance, and different spectra from older molecules (openingup multi-color control of cells).

EXAMPLES Example 1

Studies were performed to prepare sequences and to expresslight-activated ion channels in cells, tissues, and subjects.Identifications and amino acid sequences of some of the light-activatedion channels in the examples are ChR88 (SEQ ID NO:2); ChR90 (SEQ IDNO:7); ChR87 (SEQ ID NO:11); ChR62 (SEQ ID NO:14), ChR93 (SEQ ID NO: 16)and ChR2 (SEQ ID NO:19), ChR88 K176R (SEQ ID NO:5). Non-limitingexemplary methods are set forth Example 1. General methods alsoapplicable to light-activated channel molecules and methods for theiruse are disclosed in publications such as US Published Application No.2010/0234273, US Published Application No. 20110165681, Chow B Y, et.al. Methods Enzymol. 2011; 497:425-43; Chow, B Y, et al. Nature 2010Jan. 7; 463(7277):98-102, the content of each of which is incorporatedby reference herein.

Studies were performed to prepare sequences and to expresslight-activated ion channels in cells, tissues, and subjects.Non-limiting exemplary methods are set forth below.

(a) In Utero Electroporation

All procedures were in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals and approved by theMassachusetts Institute of Technology Committee on Animal Care. C57BL/6JE16-timed pregnant mice were used for electroporation. Surgery was doneunder ketamine-xylazine anesthesia and buprenorphine analgesia, DNAsolution containing plasmids of interest were injected into lateralventricle of each embryo using a pulled capillary tube. Five squarepulses (50 ms width, 1 Hz, 35V) were applied using tweezer electrode forelectroporation.

(b) Slice Preparation

P20-P30 mice were used for slice preparation. In younger animals it wasdifficult to elicit synaptic responses by photostimulating callosalaxons. Mice were anesthetized with isofluorane and transcardialyperfused with artificial cerebrospinal fluid (ACSF). The brain wasremoved and placed in an ice-cold cutting solution containing 110 mMcholine chloride, 25 mM NaHCO₃, 25 mM D-glucose, 11.6 mM sodiumascorbate, 7 mM MgCl₂, 3.1 mM sodium pyruvate, 2.5 mM KCl, 1.25 mMNaH₂PO₄ and 0.5 mM CaCl₂. 300-mm-thick coronal slices of the visualcortex were cut with a vibrating slicer and incubated in oxygenated ACSFfor 45 min at 37° C. before the recordings.

(c) Slice Electrophysiology

Recordings were performed at room temperature (22-24° C.) under constantperfusion of oxygenated ACSF. Neurons were visualized using infrareddifferential interference optics and patched with borosilicate pipettes(resistance 4-6 MO). The intracellular solution contained 120 mMpotassium gluconate, 5 mM NaCl, 2 mM MgCl₂, 0.1 mM CaCl₂, 10 mM HEPES,1.1 mM EGTA, 4 mM magnesium ATP, 0.4 mM disodium GTP, (pH 7.25; 290mOsm). Cells were recorded at a depth of 30-120 um in the brain slice.Photostimulation was done using a blue LED (470 nm; Thorlabs, Newton,N.J.) and a red LED (625 nm with 632/22 nm filter; Thorlabs).

(d) Neuron Culture, Transfection, Infection, and Imaging

All procedures involving animals were in accordance with the NationalInstitutes of Health Guide for the care and use of laboratory animalsand approved by the Massachusetts Institute of Technology Animal Careand Use Committee. Swiss Webster or C57 mice [Taconic (Hudson, N.Y.) orJackson Labs (Bar Harbor, Me.)] were used. For hippocampal cultures,hippocampal regions of postnatal day 0 or day 1 mice were isolated anddigested with trypsin (1 mg/ml) for ˜12 min, and then treated with Hankssolution supplemented with 10-20% fetal bovine serum and trypsininhibitor (Sigma Aldrich, St Louis, Mo.). Tissue was then mechanicallydissociated with Pasteur pipettes, and centrifuged at 1000 rpm at 4° C.for 10 min. Dissociated neurons were plated at a density ofapproximately four hippocampi per 20 glass coverslips, coated withMatrigel (BD Biosciences, San Jose, Calif.). For cortical cultures,dissociated mouse cortical neurons (postnatal day 0 or 1) were preparedas previously described, and plated at a density of 100-200 k per glasscoverslip coated with Matrigel (BD Biosciences). Cultures weremaintained in Neurobasal Medium supplemented with B27 (Invitrogen [LifeTechnologies, Grand Isle, N.Y.]) and glutamine. Hippocampal and corticalcultures were used interchangeably; no differences in reagentperformance were noted.

Neurons were transfected at 3-5 days in vitro using calcium phosphate(Invitrogen). GFP fluorescence was used to identify successfullytransfected neurons. Alternatively, neurons were infected with 0.1-3 μlof lentivirus or adeno-associated virus (AAV) per well at 3-5 days invitro.

(e) HEK 293FT Cell Culture and Transfection

HEK 293FT cells (Invitrogen) were maintained between 10-70% confluencein D10 medium (Cellgro [Mediatech/Corning, Manassas, Va.]) supplementedwith 10% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin(Cellgro), and 1% sodium pyruvate (Biowhittaker, Walkersville, Md.). Forrecording, cells were plated at 5-20% confluence on glass coverslipscoated with Matrigel (BD Biosciences). Adherent cells were transfectedapproximately 24 hours post-plating either with TransLT 293lipofectamine transfection kits (Minis Bio, LLC, Madison, Wis.) or withcalcium phosphate transfection kits (Invitrogen), and recorded viawhole-cell patch clamp between 36-72 hours post-transfection.

(f) In Vitro Whole Cell Patch Clamp Recording & Optical Stimulation

Whole cell patch clamp recordings were made using a Multiclamp 700Bamplifier, a Digidata 1440 digitizer, and a PC running pClamp (MolecularDevices). Neurons were bathed in room temperature Tyrode containing 125mM NaCl, 2 mM KCl, 3 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 30 mM glucose,0.01 mM NBQX and 0.01 mM GABAzine. The Tyrode pH was adjusted to 7.3with NaOH and the osmolarity was adjusted to 300 mOsm with sucrose. HEKcells were bathed in a Tyrode bath solution identical to that forneurons, but lacking GABAzine and NBQX. Borosilicate glass pipettes(Warner Instruments, Hamden, Conn.) with an outer diameter of 1.2 mm anda wall thickness of 0.255 mm were pulled to a resistance of 3-9 MΩ witha P-97 Flaming/Brown micropipette puller (Sutter Instruments, Novato,Calif.) and filled with a solution containing 125 mM K-gluconate, 8 mMNaCl, 0.1 mM CaCl₂, 0.6 mM MgCl₂, 1 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP,and 0.4 mM Na-GTP. The pipette solution pH was adjusted to 7.3 with KOHand the osmolarity was adjusted to 298 mOsm with sucrose. Accessresistance was 5-30 MΩ, monitored throughout the voltage-clamprecording. Resting membrane potential was ˜−60 mV for neurons and ˜−30mV for HEK 293FT cells in current-clamp recording.

Photocurrents were measured with 500 ms light pulses in neuronsvoltage-clamped at −60 mV, and in HEK 293FT cells voltage-clamped at −30mV. Light-induced membrane hyperpolarizations were measured with 500 mslight pulses in cells current-clamped at their resting membranepotential. Light pulses for all wavelengths except 660 nm and actionspectrum characterization experiments were delivered with a DG-4 opticalswitch with 300 W xenon lamp (Sutter Instruments), controlled via TTLpulses generated through a Digidata signal generator. Green light wasdelivered with a 575±25 nm bandpass filter (Chroma) and a 575±7.5 nmbandpass filter (Chroma Technology Group, Bellows Falls, Vt.). Actionspectra were taken with a Till Photonics Polychrome V, 150 W Xenon lamp,15 nm monochromator bandwidth.

Data was analyzed using Clampfit (Molecular Devices) and MATLAB(Mathworks, Inc.)

(g) Ion Conductance Recording

Whole-cell patch clamp recordings were performed in isolated HEK293FTcells to accurately measure parameters from single cells. All recordingswere performed using an Axopatch 200B amplifier and Digidata 1440digitizer (Molecular Devices) at room temperature. In order to allowisolated cell recording, cells were plated at a lower density of 15,000cells per well in 24-well plates that contained round glass coverslips(0.15 mm thick, 25 mm in diameter, coated with 2% Growth Factor ReducedMatrigel in DMEM for 1 h at 37° C.). For most recordings, Tyrode wasused as the extracellular solution, and the intracellular solutionconsisted of (in mM) 125 K-Gluconate, 8 NaCl, 0.1 CaCl₂, 0.6 MgCl₂, 1EGTA, 10 HEPES, 4 MgATP, 0.4 NaGTP, pH 7.3 (KOH adjusted), with 295-300mOsm (sucrose adjusted). Extracellular and intracellular solutions usedfor testing ion permeability are listed in Table 1.

TABLE 1 Compositions of solutions used in ion permeability experiments[Na] [K] [Ca] [H] Solution (mM) (mM) (mM) (mM) pH Other Intracellular 0140 0 5.10E−05 7.4 5 mM EGTA, 2 mM MgCl2, 10 mM HEPES 145 mM 145 5 15.10E−05 7.4 10 mM HEPES, NaCl 5 mM glucose, 2 mM MgCl2 145 mM 0 145 15.10E−05 7.4 10 mM HEPES, KCl 5 mM glucose, 2 mM MgCl2 90 mM 0 5 915.10E−05 7.4 10 mM HEPES, CaCl₂ 5 mM glucose, 2 mM MgCl2 5 mM 5 5 15.10E−04 6.4 135 mM NMDG, NaCl 10 mM HEPES, 5 mM glucose, 2 mM MgCl2

Liquid junction potentials were measured using standard procedures to be5.8 mV for the 90 mM CaCl₂ and 4.9 mV for the 5 mM NaCl extracellularsolutions, which were corrected during recording; the others were <1 mVin junction potential.

In all patch clamp recordings, a stringent cutoff of access resistanceless than 25 MS) and holding current less than ±50 pA was applied inorder to ensure accurate measurement. Typical membrane resistance wasbetween 500 MΩ-2 GΩ and pipette resistance was between 4-10 MΩ.

Photostimulation of patch clamped cells was conducted by a 470 nm LED(Thorlabs) at 10 mW/mm² unless otherwise stated. For most experiments,is illumination was delivered to measure transient and steady-statephotocurrents.

(h) Plasmid Construction and Site Directed Mutagenesis.

Opsins were mammalian codon-optimized, and synthesized by Genscript(Genscript Corp., NJ). Opsins were fused in frame, without stop codons,ahead of GFP (using BamHI and Agel) in a lentiviral vector containingthe CaMKII promoter, enabling direct neuron transfection, HEK celltransfection (expression in HEK cells is enabled by a ubiquitouspromoter upstream of the lentiviral cassette), and lentivirus productionand transfection. Amino acid sequences of some opsins that were testedwere as follows: ChR88 (SEQ ID NO:2); ChR90 (SEQ ID NO:7); ChR87 (SEQ IDNO:11); ChR62 (SEQ ID NO:14), ChR93 (SEQ ID NO: 16) and ChR2 (SEQ IDNO:19), ChR88 K176R (SEQ ID NO:5).

The ss' signal sequence from truncated MHC class I antigen correspondedto amino acid sequence (M)VPCTLLLLLAAALAPTQTRA (SEQ ID NO:21), DNAsequence gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc(SEQ ID NO:20). The ‘ER2’ ER export sequence corresponded to amino acidsequence FCYENEV (SEQ ID NO:23), DNA sequence ttctgctacgagaatgaagtg (SEQID NO:22). The ‘KGC’ signal sequence corresponded to amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:25), DNA sequence of KGC signalsequence: aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt.(SEQ ID NO:24).

Point mutants for HEK cell testing were generated using the QuikChangekit [Stratagene, (Agilent Technologies, Santa Clara, Calif.)] on theopsin-GFP fusion gene inserted between BamHI and Agel sites in amodified version of the pEGFP-N3 backbone [Invitrogen, (LifeTechnologies Corporation, Carlsbad, Calif.)]. All constructs wereverified by sequencing.

(i) Lentivirus Preparation

HEK293FT cells [Invitrogen, (Life Technologies Corporation, Carlsbad,Calif.)] were transfected with the lentiviral plasmid, the viral helperplasmid Δ8.74, and the pseudotyping plasmid pMD2.G. The supernatant oftransfected HEK cells containing virus was then collected 48 hours aftertransfection, purified, and then pelleted through ultracentrifugation.Lentivirus pellet was resuspended in phosphate buffered saline (PBS) andstored at −80° C. until further usage in vitro or in vivo. The estimatedfinal titer is approximately 10⁹ infectious units/mL.

Example 2

Light-activated ion channels VChR1, ChR1, ChR2, ChR87, ChR90, and ChR88were expressed in cultured hippocampal neurons using neuron culture,transfection, infection, and imaging methods described in Example 1. Invitro whole cell patch claim recording and optical stimulation wereconducted on the neurons using methods described in Example 1. FIG. 1shows channelrhodopsin photocurrents measured in the culturedhippocampal neurons. FIG. 1A shows results using red light (660 nm) peakphotocurrent at 10 mW mm⁻² for 1 s illumination. ChR88 is the only redlight sensitive channelrhodopsin with significant photocurrent at 660nm. FIG. 1B shows results using blue (4.23 mW mm⁻²) or green (3.66 mWmm⁻²) light peak photocurrent at equal photon flux for 5 msillumination. ChR87, ChR88, and ChR90 all have greater or comparablephotocurrent than ChR2. Solid bar indicates blue light, horizontalstriped bar indicates green light.

Example 3

HEK 293FT cells were transfected to express ChR2, ChR90, VChR1, ChR88,and ChR87 light-activated ion channels using methods of Example 1. Invitro whole cell patch claim recording and optical stimulation wereconducted on the transfected, cultured cells using methods described inExample 1. FIG. 2 show action spectrum at equal photon dose at allwavelengths recorded in HEK293FT cells. ChR2 (470 nm peak) and VChR1(545 nm peak) represent the existing channelrhodopsin color sensitivityrange. ChR87 (515 nm peak) and ChR90 (500 nm peak) are blue green lightsensitive channelrhodopsins. Whereas ChR88 (590 nm peak) is the firstred light sensitive natural channelrhodopsin.

Example 4

Light-activated ion channels ChR90 and ChR88 were expressed in culturedhippocampal neurons using neuron culture, transfection, infection, andimaging methods described in Example 1. In vitro whole cell patch claimrecording and optical stimulation were conducted on the neurons usingmethods described in Example 1. FIG. 3 shows optically-driven spikes inthe cultured hippocampal neurons. FIG. 3A shows red-light-driven spiketrains at low frequency for Ch88. Generally ChR88 could reliably drivespikes up to 5 Hz. However at higher frequency such as 20 Hz, ChR88desensitizes and/or causes depolarization block. FIG. 3B showsgreen-light-driven spike trains at high frequency for Ch90. Due to ChR90fast tau off and peak photocurrent recovery kinetics, it was able todrive temporally precise spikes at the highest frequency a neuron iscapable of mediating.

Example 5

Light-activated ion channels ChR88, Chr2, ChR87, and ChR90 wereexpressed in cultured hippocampal neurons using neuron culture,transfection, infection, and imaging methods described in Example 1. Invitro whole cell patch claim recording and optical stimulation wereconducted on the neurons using methods described in Example 1. FIG. 4illustrates the results and shows channelrhodopsin kinetics measured inthe hippocampal neuron culture voltage clamped at −65 mV. FIG. 4A showssingle exponential channel turn-off kinetics based on 5 ms pulse. ChR90had the fastest turn-off kinetics (3.5 ms) observed across all naturalchannelrhodopsins. FIG. 4B showed peak photocurrent recovery ratio basedon 1 s illumination. ChR87 and ChR90 both had fast peak photocurrentrecovery at around 70%. However ChR88 had slow recovery kinetics similarto ChR2.

Example 6

Light-activated ion channels Chrimson (ChR88) were expressed in culturedhippocampal neurons using neuron culture, transfection, infection, andimaging methods described in Example 1. In vitro whole cell patch claimrecording and optical stimulation were conducted on the neurons usingmethods described in Example 1. FIG. 5 shows Chrimson blue lightcrosstalk characterization in cultured neurons. FIG. 5A shows actionspectrum of Chrimson and the blue light (470 nm) wavelength used forillumination. Wavelength was chosen to minimize crosstalk. FIG. 5B showsrepresentative traces from a single neuron at various illuminationconditions. When the blue light power was doubled from 0.1 to 0.2 mWmm⁻² while the stimulation protocol was fixed as 5 ms 5 Hz, the voltagedeflection was also doubled. However when the blue light power was fixedat 0.1 mW mm⁻² but the pulse duration was changed from 5 ms to 1000 ms,the crosstalk was changed from <5 mV to full spiking correspondingly.This means blue light crosstalk was a function of both light power andlight pulse duration (total photon count).

Example 7

Light-activated ion channels Chronos (ChR90) and ChR2 were expressed incultured hippocampal neurons using neuron culture, transfection,infection, and imaging methods described in Example 1. In vitro wholecell patch claim recording and optical stimulation were conducted on theneurons using methods described in Example 1. FIG. 6 illustrates Chronosand ChR2 blue light sensitivity in cultured hippocampal neurons. FIG. 6Ais a spike irradiance curve for individual neurons. FIG. 6B shows lowestlight power needed for single-cell 100% spike probability vs GFPfluorescence. Chronos (circles) was approximately 5 times more lightsensitive than ChR2 (triangles) at a given (GFP) expression level. FIG.6C shows example traces of Chronos spiking at various light powers. FIG.6D illustrates that controls showed no significant electricaldifferences between ChR2 and Chronos expressing neurons.

Example 8

In utero electroporation and slice preparation methods as described inExample 1 were used to examine Chronos (ChR90) and Chrimson (ChR88)activation. FIG. 7 illustrates the strategy used for slicecharacterization of Chronos and Chrimson. FIG. 7A shows illuminationwavelength used for slice experiments. FIG. 7B provides micrographicimages showing histology for Chronos and Chrimson GFP fusion constructsingly expressed in layer 2/3 visual cortex in mice.

Example 9

In utero electroporation, slice preparation, and slice electrophysiologymethods as described in Example 1 were used to characterize Chrimson(ChR88) and Chronos (ChR90) blue and red light sensitivity in slicepreparations. FIG. 8 illustrates results obtained using whole cell patchclamp methods. FIG. 8A shows that red light elicited 100% spiking inChrimson expressing neurons but not Chronos expressing neurons between1-6.5 mW mm⁻². FIG. 8B shows that blue light at 0.2-0.5 mW mm⁻² couldelicit 100% spiking in Chronos expressing cells but not Chrimsonexpressing cells. However full spiking crosstalk in Chrimson expressingcells can occur at powers higher than 0.6 mW mm⁻². FIG. 8C shows bluelight crosstalk voltage of Chrimson expressing neurons.

Example 10

In utero electroporation, slice preparation, and slice electrophysiologymethods as described in Example 1 were used to characterize Chrimson(ChR88) and Chronos (ChR90). FIG. 9 illustrates results with exampletraces of current-clamped opsin-expressing neurons in layer 2/3 sliceblue light 0.1 mW mm⁻², red light 1 mW mm⁻² expressing. No crosstalk wasobserved under red light for Chronos while minimal subthreshold (<5 mV)crosstalk was observed under blue light for Chrimson.

FIG. 10 illustrates results with example traces of voltage-clampednon-opsin-expressing neurons in layer 2/3 or 5, post-synaptic toopsin-expressing cells. Zero post-synaptic crosstalk was observed forboth Chronos and Chrimson under red and blue light illuminationrespectively. Chronos: blue light 0.13 mW mm⁻², red light 1.7 mW mm⁻².Chrimson: blue light 0.37 mW mm⁻², red light 1.7 mW mm⁻².

FIG. 11 illustrates results of studies of paired-pulse illumination inslice that differentially express Chrimson and Chronos in separateneurons. FIG. 11A shows a triple plasmid in utero electroporation schemeused to obtain non-overlapping expression of Chrimson and Chronos. FIG.11B shows opsin expression in visual cortex no overlap of GFP and m02was observed ratio of Chronos to Chrimson labeling could be tuned bytitrating Cre plasmid. FIG. 11C shows voltage-clampednon-opsin-expressing neuron in layer 2/3 paired-pulse stimulation todemonstrate different synapses were selectively driven by blue and redlight. blue: 0.2 mW mm⁻²; red: 5 mW mm⁻².

Example 11

Chrimson light-activated ion channels (ChR88) were expressed in culturedhippocampal neurons using neuron culture, transfection, infection, andimaging methods described in Example 1. In vitro whole cell patch claimrecording and optical stimulation were conducted on the neurons usingmethods described in Example 1. FIG. 12 illustrated results that showedthat Chrimson could drive spikes in the far-red (660 nm) using 5 mspulses at 2.6 mW mm⁻² in cultured hippocampal neurons.

Example 12

Substituted Chrimson (ChR88) light activated ion channels, referred toas “ChR88 K176R”, having an amino acid sequence set forth as SEQ IDNO:5, were expressed in cultured hippocampal neurons using neuronculture, transfection, infection, and imaging methods described inExample 1. In vitro whole cell patch claim recording and opticalstimulation were conducted on the neurons using methods described inExample 1. FIG. 13 show results indicating that the ChR88 K176R mutanthad improved kinetics (13 ms tau off) and could mediate high frequencyspikes in cultured hippocampal neurons. Examples of current clampedtraces of a single ChR88 K176R expressing neuron are shown. FIG. 13Ashows that ChR88 K176R could reliably drive spikes from 1 to 10 mW mm⁻²at 625 nm 5 Hz stimulation. FIG. 13B shows red light (625 nm) drivenspike trains at various frequencies for ChR88 K176R. 1 mW mm⁻² lightpower is used for all frequencies. FIG. 13C shows current injectioncontrol that demonstrated that the neuron was capable of spiking at theindicated frequencies.

Example 13

Ion conductance recording methods set forth in Example 1, were used toexamine channel closing kinetics for ChR88, ChR90, ChR87, and ChR2expressed in HEK293 cells. The closing kinetics were examined andcompared. 2 ms light pulse was used to activate channelrhodopsin and allmeasurements were voltage clamped to −65 mV. Chronos had the fastestchannel closing kinetics and is independent of voltage.

Example 14

Genes described under (a), (b) and (c) were expressed in cells usingmethods provided below.

Genes

a) The Chloromonas subdivisa gene referred to herein as ChR87 and havingthe amino acid sequence set forth herein as SEQ ID NO:5 and a mammaliancodon-optimized DNA sequence set forth herein as SEQ ID NO:6;b) The gene for Chlamydomonas noctigama referred to herein as ChR88 orChrimson, and having the amino acid sequence set forth herein as SEQ IDNO:1 and a mammalian codon-optimized DNA sequence set forth herein asSEQ ID NO:2; andc) The gene for Stigeoclonium helveticum, referred to herein as ChR90 orChronos and having the amino acid sequence set forth herein as SEQ IDNO:3 and having a mammalian codon-optimized DNA sequence set forthherein as SEQ ID NO:4 are expressed in cells as follows.

Methods

(1) The opsin gene was cloned into a lentiviral or adeno-associatedvirus (AAV) packaging plasmid, or another desired expression plasmid,and then clone GFP downstream of the preferred gene, eliminating thestop codon of the opsin gene, thus creating a fusion protein.(2) The viral or expression plasmid contained either a strong generalpromoter, a cell-specific promoter, or a strong general promoterfollowed by one more logical elements (such as a lox-stop-lox sequence,which will be removed by Cre recombinase selectively expressed in cellsin a transgenic animal, or in a second virus, thus enabling the stronggeneral promoter to then drive the gene.(3) If using a viral plasmid, synthesize the viral vector using theviral plasmid.(4) If using a virus, as appropriate for gene therapy (over 600 peoplehave been treated with AAV carrying various genetic payloads to date, in48 separate clinical trials, without a single adverse event), inject thevirus using a small needle or cannula into the area of interest, thusdelivering the gene encoding the opsin fusion protein into the cells ofinterest. If using another expression vector, directly electroporate orinject that vector into the cell or organism (for acutely expressing theopsin, or making a cell line, or a transgenic mouse or other animal).(5) Illuminate with light. For Chronos, peak illumination wavelengthsare 500 nm+/−15 nm. For Chrimson, peak illumination wavelengths are 590nm+/−15 nm.(6) To illuminate two different populations of cells (e.g., in a singletissue) with two different colors of light, first target one populationwith Chrimson, and the other population with Chronos, using twodifferent viruses (e.g., with different coat proteins or promoters) ortwo different plasmids (e.g., with two different promoters). Then, afterthe molecule expresses, illuminate the tissue with 470±10 nm or 406±10nm light to preferentially depolarize the Chronos-expressing cells, andilluminate the tissue with 406±10 nm or 660±10 nm light, topreferentially depolarize the Chrimson-expressing cells.(7) The above wavelengths illustrate typical modes of operation, but arenot meant to constrain the protocols that can be used. Either narroweror broader wavelengths, or differently-centered illumination spectra,can be used. For prosthetic uses, the devices used to deliver light maybe implanted. For drug screening, a xenon lamp or LED can be used todeliver the light.

Aspects of the invention include compositions of matter that have beenreduced to practice, as described below:

Plasmids encoding for the above genes, have been prepared and used todeliver genes into cells, where the genes have been expressed. As anexemplary vector, lentiviruses carrying payloads encoding for the abovegenes have been prepared and used to deliver genes into cells resultingin expression of the light activated ion channels in the cells. Inaddition, adeno-associated viruses carrying payloads encoding for theabove genes have been prepared and used to deliver genes into cells,resulting in the expression of the light activated ion channels in thecells. Cells have been prepared that express the light activated ionchannels genes set forth in Example 2. Animals have been prepared thatinclude cells that express the light activated ion channels genesdisclosed herein.

Example 15

Two-color assays are performed. Chronos (for blue light activation) andChrimson (for red light activation) are expressed in separate sets cellsthat represent non-overlapping neuronal populations. Followingexpression, the cell population is exposed to light and the wavelengthand timing and “dose” of light is optimized using the followingparameters.

The population is contacted with blue light having a wavelength between450 nm to 500 nm, and with a pulse width of between 1 and 5 ms foractivation. A pulse width of 5 ms provides for minimum sub-thresholdcrosstalk in the blue light, which is defined as <15 mV, <10 mV, andoptimally as <5 mV.

(1) The maximum blue light power that can be used is determined using bypatch clamping Chrimson expressing cells, illuminating with blue lightand measuring voltage deflection. Optimally using blue light power suchthat maximum voltage deflection is <10 mV, usually 0.4 to 0.6 mW/mm².(2) The optimal blue light power that can be used to drive Chronos isdetermined using the same conditions as above in (1), except using lowerlight power, such as 50 μW/mm² to 0.4 mW/mm², and optimally 0.2 mW/mm².Power depends on expression system and cell type used in the study.

The population is contacted with red light having a wavelength between620 nm to 640 nm, and with a pulse width of between 1 and 5 ms foractivation, which may be optimized at 5 ms. The optimal light power todrive Chrimson in the red is determined by ramping light powers from 0.5mW/mm² to 10 mW/mm². The method is optimized such that a minimum redlight power is used to achieve 100% spiking for Chrimson.

It is to be understood that the methods, compositions, and apparatuswhich have been described above are merely illustrative applications ofthe principles of the invention. Numerous modifications may be made bythose skilled in the art without departing from the scope of theinvention.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety.

We claim:
 1. A method of altering ion conductivity of a membrane, the method comprising, expressing in a membrane in a cell a light-activated ion channel polypeptide encoded in a recombinant nucleic acid vector comprising a heterologous nucleic acid molecule insert encoding a light-activated ion channel amino acid polypeptide sequence, the amino acid sequence having at least 90% amino acid identity to amino acids 86-320 as set forth in SEQ ID NO: 2 and at least 95% identity to the amino acids 321-350 as set forth as SEQ ID NO: 2, wherein the light-activated ion channel polypeptide is expressed as part of a heterologous fusion protein, and contacting the expressed light-activated ion channel polypeptide with a light that activates the light-activated ion channel polypeptide and alters the ion conductivity of the membrane.
 2. The method of claim 1, wherein the cell is a mammalian cell.
 3. The method of claim 1, wherein the cell is an excitable cell.
 4. The method of claim 1, wherein altering the ion conductivity of the membrane comprises depolarizing the membrane.
 5. The method of claim 1, further comprising contacting the membrane comprising the light-activated ion channel polypeptide with light under conditions suitable for altering ion conductivity of the membrane; contacting the membrane with a candidate compound; and identifying the presence or absence of a change in ion conductivity of the membrane contacted with the light and the candidate compound compared to an ion conductivity in a control cell membrane comprising the expressed light-activated ion channel polypeptide and contacted with the light and not contacted with the candidate compound; wherein a change in the ion conductivity in the membrane compared to the control ion conductivity indicates an effect of the candidate compound on the ion conductivity of the membrane.
 6. The method of claim 5, wherein the cell is a mammalian cell.
 7. The method of claim 5, wherein the cell is an excitable cell.
 8. The method of claim 5, wherein the effect of the candidate compound on the ion conductivity of the membrane is depolarization of the test membrane.
 9. A method of assessing the effect of a candidate compound on ion conductivity of a membrane, the method comprising, (a) expressing in a membrane in a cell a light-activated ion channel polypeptide encoded in a recombinant nucleic acid vector comprising a heterologous nucleic acid molecule insert encoding a light-activated ion channel amino acid polypeptide sequence, the amino acid sequence having at least 90% amino acid identity to amino acids 86-320 as set forth in SEQ ID NO: 2 and at least 95% identity to the amino acids 321-350 as set forth as SEQ ID NO: 2, wherein the light-activated ion channel polypeptide is expressed as part of a heterologous fusion protein; (b) contacting the membrane comprising the light-activated ion channel polypeptide with light under conditions suitable for altering ion conductivity of the membrane; (c) contacting the membrane with a candidate compound; and (d) identifying the presence or absence of a change in ion conductivity of the membrane contacted with the light and the candidate compound compared to an ion conductivity in a control cell membrane comprising the expressed light-activated ion channel polypeptide and contacted with the light and not contacted with the candidate compound; wherein a change in the ion conductivity in the membrane compared to the control ion conductivity indicates an effect of the candidate compound on the ion conductivity of the membrane.
 10. The method of claim 9, wherein the cell is a mammalian cell.
 11. The method of claim 9, wherein the cell is an excitable cell.
 12. The method of claim 9, wherein the effect of the candidate compound on the ion conductivity of the membrane is depolarization of the test membrane. 